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International audience; The effects of two pretreatments (microwaves or oven-drying) on the dogfish (Squalus acanthias) skin as well as two drying processes (freeze-drying or spray-drying) on the extracted gelatins were studied. Thus six types of gelatins were obtained, three of which were freeze-dried (FG) and the others were spray-dried (SG), from the untreated skin (US), microwaves-pretreated skin (MS) and oven-pretreated skin (OS). The highest yield (8.67%) was obtained for the OSFG, while the lowest one (3.06%) was measured for the OSSG. Interestingly, all gelatins exhibited relatively high protein (84.02-89.53%), and low lipid (0.50-1.71%) and ash (3.05-7.17%) contents. In addition, gelatins were analyzed by the Fourier transform infrared and the spectra displayed important differences in some specific peaks, particularly in the amide I, amide II and amide III. The gelatins extracted from the untreated skin, regardless the drying method, presented the highest foaming capacity. The textural profile analysis showed that USSG was the hardest (213.6 g) and the chewier (23.8 N × mm) gelatin. Moreover, analysis of thermal properties showed that USSG also has the highest glass-transition temperature. The interesting properties of gelatin extracted from dogfish skin encourage their future use as a functional ingredient in industrial food formulations.

The transport mechanisms for water, ammonia and urea in elasmobranch gill, kidney and gastrointestinal tract remain to be fully elucidated. Aquaporin 8 (AQP8) is a known water, ammonia and urea channel that is expressed in the kidney and respiratory and gastrointestinal tracts of mammals and teleost fish. However, at the initiation of this study in late 2019, there was no copy of an elasmobranch aquaporin 8 gene identified in the genebank even for closely related holocephalon species such as elephant fish (Callorhinchus milii) or for the elasmobranch little skate (Leucoraja erinacea). A transcriptomic study in spiny dogfish (Squalus acanthias) also failed to identify a copy. Hence this study has remedied this and identified the AQP8 cDNA sequence using degenerate PCR. Agarose electrophoresis of degenerate PCR reactions from dogfish tissues showed a strong band from brain cDNA and faint bands of a similar size in gill and liver. 5' and 3' RACE was used to complete the AQP8 cDNA sequence. Primers were then designed for further PCR reactions to determine the distribution of AQP8 mRNA expression in dogfish tissues. This showed that AQP8 is only expressed in dogfish brain and AQP8 therefore clearly can play no role in water, ammonia and urea transport in the gill, kidney or gastrointestinal tract. The role of AQP8 in dogfish brain remains to be determined.

Nitrogen recycling through the gut microbiome is an important mechanism used throughout vertebrates to reclaim valuable nitrogen trapped in urea. Evidence suggests it may be especially important in nitrogen limited animals, yet little is known about its role in marine elasmobranchs, which are said to be severely nitrogen limited. In the present study we used antibiotics to deplete the gut microbiome of Pacific spiny dogfish and assessed the role of the microbiome in nitrogen handling in both fed and fasted states. In fed animals, antibiotic treatment eliminated the activity of the microbial enzyme urease and reduced cellulase activity by 78%. This reduction in microbial enzyme activity resulted in significantly lower plasma urea levels which then trended upward as urea excretion rates decreased. Ammonia excretion rates were also significantly lower in antibiotic treated fish compared to the control fed. Finally, antibiotic treated fed individuals lost an average of 7.4% of their body mass while the fed controls lost only 1.8% of their body mass. Nitrogen handling in fasted animals was not significantly impacted by a reduction in microbial activity. These results suggest that compromising the gut microbiome significantly influences post-prandial nitrogen handling in spiny dogfish, and that the recycling of urea‑nitrogen may be vital to maintaining nitrogen balance in these fish.

Three aquaporin water channel proteins, AQP3, AQP4 and AQP15 were localized to cells within the kidney of the spiny dogfish, Squalus acanthias, using an immunohistochemical approach. Dogfish kidney has two zones, the bundle zone (including five nephron segment bundles) and the sinus zone (with two major loops). In order to discriminate between the two loops, the cilia occurring in the first proximal/intermediate loop were labeled with two antibodies including an anti-acetylated tubulin antibody. The second late distal tubule loop (LDT) was identified, as the nephron in that region has no luminal cilia. Strong staining of the rabbit anti-dogfish AQP3, AQP4 (AQP4/2) or AQP15 polyclonal antibodies localized to LDT tubules. These antibodies were further co-stained with a mouse anti-Na+,K+-ATPase a5 monoclonal antibody, as Na+,K+-ATPase has previously been suggested to localize to the early distal tubule (EDT) and LDT and a mouse anti-NKCC T4 antibody, as NKCC2 was previously suggested to be located in the EDT and the second half of the LDT. In the LDT, strong AQP4/2 and AQP15 antibody staining localized together with the strong Na+,K+-ATPase antibody staining, whereas strong AQP3 antibody staining was largely separate but with an overlapping distribution. Very low levels of AQP4/2 antibody basal membrane staining was also detected in the first proximal /intermediate loop of the sinus zone. There was no mouse anti-NKCC T4 antibody staining apparent in the LDT. In the convoluted part of the bundle zone, the AQP4/2 and Na+,K+-ATPase but not the AQP3 or AQP15 antibodies stained tubule segments, with both AQP4/2 and Na+,K+-ATPase staining the EDT, and with low-level AQP4/2 staining of two other tubules of the bundle, which were most likely to be the proximal 1a (PIa) and intermediate II (IS II) tubules. The AQP4/2 antibody also stained the EDT in the straight bundle zone. The mouse anti-NKCC T4 antibody stained the apical region of EDT tubules in the convoluted bundle zone, suggesting that the antibody was binding to the NKCC2 cotransporter. The AQP15 antibody appeared to bind to the peritubular sheath surrounding bundles in the bundle zone. Due to the AQP4/2 antibody staining in the EDT that immediately proceeds and continues into the LDT, this suggested that the strong AQP4/2, AQP15 and Na+,K+-ATPase antibody staining was located at the beginning of the LDT and therefore the strong AQP3 was located at the end of the LDT. The staining of all three AQP antibodies was blocked by the peptide-antigen used to make each one, suggesting that all the staining is specific to each antibody.

The antidiabetic potential of thirteen novel dogfish glucagon derived analogues were assessed in vitro and in acute in vivo studies. Stable peptide analogues enhanced insulin secretion from BRIN-BD11 β-cells (p

In this paper, we report the synthesis and application of enzyme-like DNA-copper oxide/platinum nanoparticles for the separate quantification of inorganic and organomercury species in various real samples. We synthesized a series of poly(thymine) (T60)–copper oxide/metal nanocomposites (T60–CuxO/M NCs; M = Au, Ag or Pt) that exhibited enzyme-like activities [oxidase (OX), peroxidase (POX), and catalase (CAT)]. The enzyme-like activities are tunable due to the incorporation of various metals into the NCs. Among a series of synthesized CuxO/M NCs, T60–copper oxide-platinum nanocomposites (T60–CuxO/Pt NCs) exhibited the highest OX-like activity via the O2-mediated oxidation of substrates, such as Amplex Red (AR), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), and 3,3′,5,5′-tetramethylbenzidine (TMB), to form fluorescent or colored products. Interestingly, inorganic mercury ions (Hg2+) and organomercury species, such as methylmercury (MeHg+), ethylmercury (EtHg+), and phenylmercury (PhHg+), significantly inhibited the OX-like activity of T60–CuxO/Pt NCs. For the selective detection of mercury species, we used ABTS in the T60–CuxO/Pt NCs system, and the ABTS/T60–CuxO/Pt NCs-based assay allowed for the detection of mercury ions at nanomolar concentrations based on the decrease in the catalytic activity caused by the mercury ions. To separately quantify the inorganic and organomercury species in a sample, we employed selenium nanoparticles (Se NPs) as a masking agent, as they preferentially bind with inorganic mercury species. The ABTS/T60–CuxO/Pt NCs-based assay with the masking agent of Se NPs further provided specificity for the detection of organomercury species in environmental water samples (tap water, river water, and seawater) and fish muscle samples (dogfish muscle DORM-II).

It is advisable to monitor and regulate inorganic arsenic (iAs) in food and feedstuff. This work describes an update and validation of a method of selective hydride generation (HG) with inductively coupled plasma mass spectrometry (ICP-MS) for high-throughput screening of iAs content in seafood samples after microwave-assisted extraction with diluted nitric acid and hydrogen peroxide. High concentration of HCl (8 M) for HG along with hydrogen peroxide in samples of a same concentration as used for extraction leads to a selective conversion of iAs to volatile arsine that is released and transported to the detector. A minor contribution from methylarsonate (≈20% to iAs) was found, while HG from dimethylarsinate, trimethylarsine oxide is substantially suppressed (less than 1% to iAs). Methodology was applied to Certified Reference Materials (CRMs) TORT-3, DORM-3, DORM-4, DOLT-4, DOLT-5, PRON-1, SQID-1 and ERM-CE278k, in some of them iAs has been determined for the first time, and to various seaweed samples from a local store. The results were always compared with a reference method and selectivity of iAs determination was evaluated. An inter-laboratory reproducibility was tested by comparative analyses of six fish and four seaweed samples in three European laboratories, with good agreement of the results. The method of HG-ICP-MS is sensitive (limit of detection 2 μg kg−1 iAs), well suited for screening of large number of samples and selective at iAs concentration levels at which maximum limits are expected to be set into EU legislation for marine samples.

The insulinotropic and antihyperglycaemic properties of glucagons from the sea lamprey (Petromyzontiformes), paddlefish (Acipenseriformes) and trout (Teleostei) and oxyntomodulin from dogfish (Elasmobranchii) and ratfish (Holocephali) were compared with those of human glucagon and GLP-1 in mammalian test systems. All fish peptides produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat and 1.1 B4 human clonal β-cells and isolated mouse islets. Paddlefish glucagon was the most potent and effective peptide. The insulinotropic activity of paddlefish glucagon was significantly (P 0.01) decreased after incubating BRIN-BD11 cells with the GLP1R antagonist, exendin-4(9-39) and the GCGR antagonist [des-His

Na(+)/H(+) Exchanger (NHE) proteins mediate cellular and systemic homeostasis of sodium and acid and may be the major sodium uptake method for fishes. We cloned and sequenced NHE2 and NHE3 from the gill of the North Pacific Spiny Dogfish shark Squalus suckleyi and expressed them in functional form in NHE-deficient (AP-1) cell lines. Estimated IC50 for inhibition of NHE activity by amiloride and EIPA were 55 μmol l(-1) and 4.8 μmol l(-1), respectively, for NHE2 and 9 μmol l(-1) and 24 μmol l(-1), respectively, for NHE3. Phenamil at 100 μmol l(-1) caused less than 16% inhibition of activity for each isoform. Although the IC50 are similar for the two isoforms, dfNHE2 is less sensitive than human NHE2 to inhibition by amiloride and EIPA, while dfNHE3 is more sensitive than human NHE3. These IC50 estimates should be considered when selecting inhibitor doses for fishes and for reinterpretation of previous studies that use these pharmacological agents.

We examined the ventilatory response of the spiny dogfish, to elevated internal or environmental ammonia. Sharks were injected via arterial catheters with ammonia solutions or their Na salt equivalents sufficient to increase plasma total ammonia concentration [ T Amm ] a by 3–5 fold from 145 ± 21 μM to 447 ± 150 μM using NH 4 HCO 3 and a maximum of 766 ± 100 μM using (NH 4 ) 2 SO 4 . (NH 4 ) 2 SO 4 caused a small increase in ventilation frequency (+14%) and a large increase in amplitude (+69%), while Na 2 SO 4 did not. However, CO 2 partial pressure ( P aCO2 ) also increased and arterial pH a and plasma bicarbonate concentration ([HCO 3 − ] a ) decreased. NH 4 HCO 3 caused a smaller increase in plasma ammonia resulting in a smaller but significant, short lived increases in ventilation frequency (+6%) and amplitude (36%), together with a rise in P aCO2 and [HCO 3 − ] a . Injection with NaHCO 3 which increased pH a and [HCO 3 − ] a did not change ventilation. Plasma ammonia concentration correlated significantly with ventilation amplitude, while ventilation frequency showed a (negative) correlation with pH a . Exposure to high environmental ammonia (1500 μM NH 4 HCO 3 ) did not induce changes in ventilation until plasma [ T Amm ] a increased and ventilation amplitude (but not frequency) increased in parallel. We conclude that internal ammonia stimulates ventilation in spiny dogfish, especially amplitude or stroke volume, while environmental ammonia only stimulates ventilation after ammonia diffuses into the bloodstream.

The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.

Nitrogen (N) appears to be a limiting dietary resource for elasmobranchs, required not only for protein growth but also for urea-based osmoregulation. Building on recent evidence that the toxicant ammonia can be taken up actively at the gills of the shark and made into the valuable osmolyte urea, we demonstrate that the uptake exhibits classic Michaelis–Menten saturation kinetics with an affinity constant (Km) of 379 µmol l−1, resulting in net N retention at environmentally realistic ammonia concentrations (100–400 µmol l−1) and net N loss through stimulated urea-N excretion at higher levels. Ammonia-N uptake rate increased or decreased with alterations in seawater pH, but the changes were much less than predicted by the associated changes in seawater PNH3, and more closely paralleled changes in seawater NH4+ concentration. Ammonia-N uptake rate was insensitive to amiloride (0.1 mmol l−1) or to a 10-fold elevation in seawater K+ concentration (to 100 mmol l−1), suggesting that the mechanism does not directly involve Na+ or K+ transporters, but was inhibited by blockade of glutamine synthetase, the enzyme that traps ammonia-N to fuel the ornithine–urea cycle. High seawater ammonia inhibited uptake of the ammonia analogue [14C]methylamine. The results suggest that branchial ammonia-N uptake may significantly supplement dietary N intake, amounting to about 31% of the nitrogen acquired from the diet. They further indicate the involvement of Rh glycoproteins (ammonia channels), which are expressed in dogfish gills, in normal ammonia-N uptake and retention.

Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity.

We analyzed Hg, Cd, Zn, Cu and Fe concentrations in liver samples as well as the Hg concentration and stable isotope ratios of carbon and nitrogen (δ13C and δ15N) in muscle samples from silvertip sharks (Carcharhinus albimarginatus) in Japan. Muscular and hepatic Hg concentrations increased with increased body length. However, these increases were more prominent in the liver than in the muscle samples, and appeared to occur after maturation. Hepatic Zn and Cu concentrations decreased during the growth stage, and then increased concomitantly thereafter with increases in Cd burden. Hepatic Fe concentration from males increased proportionally with increases in body length, whereas no increase was observed in samples from females, probably due to the mother-to-embryo transfer of Fe. The δ13C values tended to decrease with increases in body length, whereas no decrease in the δ15N values was observed.

A fast method for the determination of As, Co, Cu, Fe, Mn, Ni, Se and V in biological samples by ETV-ICP-MS, after a simple sample treatment with formic acid, is proposed. Approximately 75mg of each sample is mixed with 5mL of formic acid, kept at 90°C for 1h and then diluted with nitric acid aqueous solution to a 5% (v/v) formic acid and 1% (v/v) nitric acid final concentrations. A palladium solution was used as a chemical modifier. The instrumental conditions, such as carrier gas flow rate, RF power, pyrolysis and vaporization temperatures and argon internal flow rate during vaporization were optimized. The formic acid causes a slight decrease of the analytes signal intensities, but does not increase the signal of the mainly polyatomic ions ( 14N 35Cl +, 14N 12C +, 40Ar 12C +, 13C 37Cl +, 40Ar 36Ar +, 40Ar 35Cl +, 35Cl 16O +, 40Ar 18O +) that affect the analytes signals. The effect of charge transfer reactions, that could increase the ionization efficiency of some elements with high ionization potentials was not observed due to the elimination of most of the organic compounds during the pyrolysis step. External calibration with aqueous standard solutions containing 5% (v/v) formic acid allows the simultaneous determination of all analytes with high accuracy. The detection limits in the samples were between 0.01 (Co) and 850μgkg -1 (Fe and Se) and the precision expressed by the relative standard deviations (RSD) were between 0.1% (Mn) and 10% (Ni). Accuracy was validated by the analysis of four certified reference biological materials of animal tissues (lobster hepatopancreas, dogfish muscle, oyster tissue and bovine liver). The recommended procedure avoids plasma instability, carbon deposit on the cones and does not require sample digestion. © 2011 Elsevier B.V.. Fil: Tormen, Luciano. Universidade Federal Da Fronteira Sul; Brasil. Universidade Federal de Santa Catarina; Brasil Fil: Gil, Raul Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; Argentina Fil: Frescura, Vera L. A.. Universidade Federal de Santa Catarina; Brasil Fil: Martinez, Luis Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Química de San Luis. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Química de San Luis; Argentina Fil: Curtius, Adilson J.. Universidade Federal de Santa Catarina; Brasil

A microwave-assisted double insert multimode vapour-phase digestion method was developed for the digestion of organic samples. The experimental set-up was based on a third generation-type teflon microwave vessel, equipped with an automatic pressure regulating type vessel cover. A borosilicate glass holder insert, containing a smaller quartz sample insert, was fitted inside the vessel. Sulphuric acid was added to the holder insert as a microwave absorbing and temperature transferring liquid, which transferred heat to the sample insert (into which the sample was weighed) and charred the sample material. Oxidation of the sample material was carried out simultaneously with charring using nitric acid vapour, which was generated by the 1:1 (v/v) sulphuric acid-nitric acid mixture located in the bottom of the microwave vessel. This set-up generated high digestion efficiency, without any of the interferences normally associated with direct sulphuric acid usage. The method was used for determining the concentrations of Cd, Cr, Cu, Mn, Mo, Zn and Fe in certified organic reference materials using ICP-OES instrumentation. The certified organic reference materials were NRCC DOLT-2 dogfish liver, NIST-SRM 1577b bovine liver and IRMM VDA cadmium in polyethylene No. 001 and No. 004. The results were in good agreement with the certified values, forepart from Cd. For Cd the results were lower than the certified values due to volatilization losses. Sample materials that could not be digested by an earlier procedure were completely digested during a single-step, 30 min digestion. The tested sample materials included certified reference materials, 3-nitrobenzoic acid (3-NBA) and pike (Esox lucius) muscle. The residual carbon concentrations in the digestion solutions were below the detection limit of the TOC instrument. This type of digestion method is described here for the first time in the literature.

The dogfish egg case is a composite structure that combines mechanical tensile strength, toughness and elasticity with high permeability to small molecules and ions. Presumably, it provides both a protective and a filtering role for the egg/embryo contained within it. In this work, we performed structural studies of the Galeus melastomus egg case at two different stages of the hardening process, utilizing ATR FT-IR and FT-Raman spectroscopy. Based on these data we deduce that: (a) The G. melastomus egg case, in close analogy to that of the related species Scyliorhinus cunicula, is a complex, composite structure which consists mainly of an analogue of collagen IV. This network forming protein appears to have common secondary structural characteristics in the entire egg case. (b) The outermost layer of the non-sclerotized egg case is especially rich in tyrosine, while the innermost layer is rich in polysaccharides, presumably glycosaminoglycans, and lipids. These differences are diminished upon hardening. (c) Disulfide bonds do not appear to play a significant role in cross-linking. However, cross-links involving tyrosine residues appear to sclerotize the egg case. It is proposed that the intensity of the Raman band at ca. 1615 cm(-1), which is due to ring stretching vibrations of Tyr, might be a useful indicator of the sclerotization status of a certain proteinaceous tissue, when tyrosines are involved in sclerotization mechanisms.

A simple home-made automatic dynamic hollow fiber based liquid-liquid-liquid microextraction (AD-HF-LLLME) device was designed and constructed for the simultaneous extraction of organomercury and inorganic mercury species with the assistant of a programmable flow injection analyzer. With 18-crown-6 as the complexing reagent, mercury species including methyl-, ethyl-, phenyl- and inorganic mercury were extracted into the organic phase (chlorobenzene), and then back-extracted into the acceptor phase of 0.1% (m/v) 3-mercapto-1-propanesulfonic acid (MPS) aqueous solution. Compared with automatic static (AS)-HF-LLLME system, the extraction equilibrium of target mercury species was obtained in shorter time with higher extraction efficiency in AD-HF-LLLME system. Based on it, a new method of AD-HF-LLLME coupled with large volume sample stacking (LVSS)-capillary electrophoresis (CE)/UV detection was developed for the simultaneous analysis of methyl-, phenyl- and inorganic mercury species in biological samples and environmental water. Under the optimized conditions, AD-HF-LLLME provided high enrichment factors (EFs) of 149-253-fold within relatively short extraction equilibrium time (25min) and good precision with RSD between 3.8 and 8.1%. By combining AD-HF-LLLME with LVSS-CE/UV, EFs were magnified up to 2195-fold and the limits of detection (at S/N=3) for target mercury species were improved to be sub ppb level.

13 páginas, 3 figuras, 2 tablas.-- This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited, This work investigates the production of hyaluronic acid (H) by Streptococcus equi subsp. zooepidemicus in complex media formulated with peptones obtained from Scyliorhinus canicula viscera by-products. Initially, in batch cultures, the greatest productions were achieved using commercial media (3.03 g/L) followed by peptones from alcalase hydrolyzed viscera (2.32 g/L) and peptones from non-hydrolyzed viscera (2.26 g/L). An increase of between 12% and 15% was found in subsequent fed-batch cultures performed on waste peptones. Such organic nitrogen sources were shown to be an excellent low-cost substrate for microbial H, saving more than 50% of the nutrient costs, Isabel Rodríguez Amado was awarded with a postdoctoral contract from the Xunta de Galicia (Plan I2C, 2012). This research was funded by the projects MARMED 2011-1/164 (Atlantic Area Programme, EU), 0687_NOVOMAR_1_P (POCTEP Programme, EU) and iSEAS LIFE13 ENV/ES/000131 (LIFE+ Programme, EU). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

To understand the role of the Tigriopus japonicus Hsp20 gene, we isolated this gene from a whole body cDNA library and found two heat shock factor elements at the 5'-UTR. The transformed bacteria containing Tigriopus Hsp20 showed thermotolerance against heat shock (54 degrees C) with different ranges of time. The Tigriopus Hsp20 gene is comprised of 174 amino acid residues and shows similarity to Caenorhabditis elegans (27% identity), silkworm (24.1% identity), moth (24.1% identity), Mexican tetra (19.5% identity), zebrafish (19.5% identity), and spiny dogfish (17.2% identity) genes, but shows more similarity in the C-terminal region that contains an alpha-crystallin domain. Protein motifs such as an N-glycosylation site (67-70 NKSE) and a casein kinase II phosphorylation site were found in Tigriopus Hsp20. The genomic structure of the Tigriopus Hsp20 gene did not contain introns. To characterize the biochemical characteristics of the Tigriopus Hsp20 protein, we expressed Tigriopus Hsp20 in Escherichia coli and purified the soluble protein via 6x His-tag chromatography. To analyze the gene expression of Tigriopus Hsp20 against environmental stresses (e.g., water temperature and salinity), we performed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). On exposure to different salinities, significant change in the expression of Tigriopus Hsp20 was not observed. However, upon heat shock (30 degrees C), Tigriopus Hsp20 expression was significantly increased, but in the case of cold shock (4 or 10 degrees C), expression was likely downregulated. These findings provide a better understanding of cellular protection mechanisms against environmental stress such as heat shock.

Background Trimethylamine (TMA) is a volatile substance produced in the gut, absorbed into the blood and further metabolized by healthy individuals into trimethylamine-N-oxide (TMAO) by TMA-oxidase and then excreted in urine. Patients suffering from trimethylaminuria (TMAU) show an impaired enzymatic oxidation of TMA, excreting this amine in breath, urine and other body secretions which confers an unpleasant body odor. Methods We diagnosed a Brazilian adult male patient suspected of trimethylaminuria with a burden of choline bitartarate by monitoring the urinary excretion of TMA and TMAO by proton nuclear magnetic resonance spectroscopy (1H-NMR). Results The patient's urinalyses showed an augmented TMA (12.64±0.95 mg/l) and TMAO (88.42±0.82 mg/l) excretion 6 h after the overload test representing an oxidation capacity of 84.6%, consistent with a heterozygosis condition. Diets containing tuna fish or eggs resulted in an excretion of TMA and TMAO similar to that of the control diet. Only the diet based on dogfish, rich in TMAO, enhanced the excretion of TMA and TMAO reaching 24.65 and 1055.55 mg/l, respectively, in the 0–24 h urine sample. Conclusions It was concluded first, that the patient was not able to metabolize the dietary overload of TMA and second, that more studies are needed to substantiate foods that should be avoided, especially regarding fish, due to their high TMA precursor contents.

We report the cloning and characterization of two melanocortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis shows that these shark receptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human counterparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. The radioligand binding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity for alpha- and beta-melanocyte stimulating hormone (MSH), and slightly lower affinity for gamma-MSH when compared with the human orthologue. ACTH(1-24) has high potency in a second-messenger cAMP assay while alpha- and gamma-MSH had slightly lower potency in cells expressing the SacMC3R. We used receptor-enhanced green fluorescence protein (EGFP) fusion to show the presence of SacMC3R in plasma membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compartments of these cells hindering pharmacological characterization. The anatomical distribution of the receptors were determined using reverse transcription PCR. The results showed that the SacMC3R is expressed in the hypothalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions. This report describes the first molecular characterization of a MC3R in fish. The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago.

Although elasmobranchs are widely distributed species, commonly found on sandy, gravely or muddy bottoms, several biological aspects of their metabolism still remain poorly investigated. In this work the efficiency of antioxidant system and detoxification enzymes were investigated in the coastal spotted dogfish Scyliorhinus canicula and in the red mullet Mullus barbatus for comparison with a teleost species. Organisms were sampled during a bottom trawl survey and analyzed for the biotransformation activity (EROD), levels of metallothioneins, catalase, glutathione S-transferases and total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals. EROD activity in the elasmobranchs was more than one order of magnitude lower than in the red mullets, while similar levels of metallothioneins were measured in these species. S. canicula showed significantly lower antioxidant enzymes and a more reduced efficiency in neutralizing OH; on the other hand the scavenging capability toward ROO was comparable in S. canicula and M. barbatus.

Teleosts and elasmobranchs faced with considerable osmotic challenges living in sea water, use compensatory mechanisms to survive the loss of water (teleosts) and urea (elasmobranchs) across epithelial surfaces. We hypothesized that the gill, with a high surface area for gas exchange must have an apical membrane of exceptionally low permeability to prevent equilibration between seawater and plasma. We isolated apical membrane vesicles from the gills of Pleuronectes americanus (winter flounder) and Squalus acanthias (dogfish shark) and demonstrated approximately sixfold enrichment of the apical marker, ADPase compared to homogenate. We also isolated basolateral membranes from shark gill (enriched 2.3-fold for Na-K-ATPase) and using stopped-flow fluorometry measured membrane permeabilities to water, urea, and NH3. Apical membrane water permeabilities were similar between species and quite low (7.4 ± 0.7 × 10−4and 6.6 ± 0.8 × 10−4cm/s for shark and flounder, respectively), whereas shark basolateral membranes showed twofold higher water permeability (14 ± 2 × 10−4cm/s). Permeabilities to urea and NH3were also low in apical membranes. Because of the much lower apical to basolateral surface area we conclude that the apical membrane represents an effective barrier. However, the values we obtained were not low enough to account for low water loss (teleosts) and urea loss (elasmobranchs) measured in vivo by others. We conclude that there are other mechanisms which permit gill epithelia to serve as effective barriers. This conclusion has implications for the function of other barrier epithelia, such as the gastric mucosa, mammalian bladder, and renal thick ascending limb.

In the shark (sa), two variants of the renal Na-K-Cl cotransporter (saNKCC2A and saNKCC2F) are produced by alternative splicing of the second transmembrane domain (tm2). In mammals, these splice variants, as well as a third variant (NKCC2B), are spatially distributed along the thick ascending limb of Henle and exhibit divergent kinetic behaviors. To test whether different tm2in saNKCC2 are also associated with different kinetic phenotypes, we examined the ion dependence of86Rb influx for shark and rabbit splice variants expressed in Xenopus laevis oocytes. We found that, in both species, A forms have higher cation affinities than F forms. In regard to Cl affinity, however, the A-F difference was more pronounced in rabbit, and the relationship between transport activity and Cl concentration was not always sigmoidal. These results show that the tm2of saNKCC2 is, as in rabbit, important for Cl transport, and they suggest that the ability of the distal NKCC2-expressing segment to extract Cl from the luminal fluid differs among species. We have also found that the renal NKCC2 of distant vertebrates share similar affinities for cations. This finding points to the existence of highly conserved residues that mediate the kinetic behavior of the NKCC2 splice variants.

We report the cloning, expression, pharmacological characterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte-stimulating hormone) peptides alpha-, beta-, and gamma-MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We confirmed that this was not due to individual polymorphism and subsequently mutated the residue 'back' to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT-PCR in the optic tectum, hypothalamus, brain stem, telencephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characterization of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue distribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago.

The indanedione series of vinyl ketone spin-labelling reagents has been extended in two ways: by increasing the length of the rigid spacer between the reactive centre and the nitroxide ring, or by introducing an electrophilic substituent (that could also hinder its rotation) at the bridge head position of the nitroxide ring. Three reagents of this new series have been used to spin label the Class II thiol groups of membranous Na,K-ATPase from Squalus acanthias. With a conjugated diene spacer, the majority of spin labels are strongly held but a minor population is relatively mobile at 37 degrees C. With a conjugated triene spacer, the nitroxide is still strongly held but a portion of the label is non-covalently bound. The 4-bromo-pyrroline derivative (with short vinyl spacer) is tightly held at the attachment site, and the conventional electron paramagnetic resonance (EPR) spectra distinguish between the two enantiomeric structures which differ in their mobility at 37 degrees C. Saturation transfer EPR (ST-EPR) spectra of this label at 4 degrees C have been used to determine the dependence of the protein rotational mobility on ionic strength. Electrostatic repulsion contributes to the lateral interactions between Na,K-ATPase molecules.

In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined

Aminosterols isolated from the dogfish shark Squalus acanthias are promising therapeutic agents in the treatment of infection and cancer. One of these, MSI-1436, has been shown to possess antimicrobial activity slightly better than squalamine. In this study, a series of analogs of MSI-1436 have been synthesized from stigmasterol. The 7 alpha-hydroxy substituent of MSI-1436 was either omitted or the stereochemistry modified to the 7 beta position. Also, analogs of MSI-1436 with 24-sulfate, 24-amino, and 24-hydroxy substituents were synthesized in order to assess the importance of the side chain functional group on antimicrobial activity. All of the analogs possess significant antimicrobial activity, suggesting that substitution at C7 and C24 of the aminosterols plays a minor role in their antimicrobial potency.

Recent examination of urea flux in the intestine of the spiny dogfish shark, Squalus acanthias , has shown that feeding significantly enhances urea uptake across the intestine, and this was significantly inhibited following mucosal addition of phloretin. The present study examined potential mechanisms of urea uptake across the dogfish intestine in starved and fed dogfish. Unidirectional flux chambers were used to examine the kinetics of urea uptake, and to determine the influence of sodium, ouabain, competitive urea analogues, and phloretin on urea uptake across the gut of fed dogfish. Intestinal epithelial preparations from starved and fed dogfish were mounted in Ussing chambers to examine the effect of phloretin on bidirectional solute transport across the intestine. In the unidirectional studies, the maximum uptake rate of urea was found to be 35.3 ± 6.9 μmol.cm -2 .h -1 and K m was found to be 291.8 ± 9.6 mM in fed fish, and there was a mild inhibition of urea uptake following mucosal addition of competitive agonists. Addition of phloretin, Na-free Ringers and ouabain to the mucosal side of intestinal epithelia also led to a significant reduction in urea uptake in fed fish. In the Ussing chamber studies there was a net influx of urea in fed fish and a small insignificant efflux in starved fish. Addition of phloretin blocked urea uptake in fed fish when added to the mucosal side. Furthermore, phloretin had no effect on ion transport across the intestinal epithelia with the exception of the divalent cations, magnesium and calcium.

The aim of the present study was to evaluate the effects of endothelin-l-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg(-1)) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O2. The effects of endothelin-1 on arterial blood partial pressure of CO2 were not synchronised with the changes in O2 partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO2 partial pressure was increased transiently by approximately 1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO2 partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (approximately 0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml(-1)) eliminated the increase in CO2 partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO2 partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by approximately 10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish ( Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl 2 at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl 2 inhibited cotransport activity in a dose-dependent manner with an apparent K i of approx. 50 μM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p -chloromercuribenzoic acid and p -chloromercuriphenylsulfonic acid at 100 μM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H 2 O 2 or 1 mM dithiothreitol for 30 min at 15°C did not change cotransport activity. Transport activity was, however, reduced by 45.9±2.5% after an incubation with 3 mM N -ethylmaleimide for 20 min. Blocking free amino groups by N -hydroxysuccinimide or biotinamidocapronate- N -hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5×10 −5 M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5±7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.

A microwave heated, vapor-phase nitric acid-hydrogen peroxide digestion method for pulverized, biological sample materials was developed. Sample masses up to 200 mg were digested using calibrated quartz inserts inside first generation type, low-pressure, Teflon-PFA microwave vessels. In the first step, samples were digested in the vapor-phase for 80 min using a progressive heating pattern. Three mL of 70% nitric acid and 0.5 mL of 30% hydrogen peroxide were used as digestion reagents. In the second step, the small residue left after first step digestion was dissolved in 1.4% nitric acid or additionally with 0.5% hydrofluoric acid by heating for 15 min. The digestion method was optimized using pike (Esox lucius) muscle as a test material. The method was further optimized using three certified reference materials. Ca, Cu, Fe, Mg and Zn were determined from NIST-SRM 1577a bovine liver by ICP-AES. Cr and Ni were determined from NIST-SRM 8433 corn bran and NRCC DOLT-2 dogfish liver by GFAAS. For all elements the values obtained were close or within certified limits. Spike recoveries were between 96 to 107%. Digestion efficiency ranged from 91 to 99%.

Oxygen consumption and heat production were measured during contraction and recovery of isolated, white muscle fibres from dogfish (Scyliorhinus canicula) at 19 degrees C. The contraction period consisted of 20 isometric twitches at 3 Hz; this was followed by a recovery period of 2 h without stimulation. We tested the hypothesis that recovery is wholly oxidative (not glycolytic) in these fibres. The following features support this hypothesis. (i) The ratio of total heat produced to oxygen consumed, 451+/−34 kJ mol(−)(1) (mean +/− s.e.m., N=29), was close to that expected for either the oxidation of carbohydrate, 473 kJ mol(−)(1), or the oxidation of fat, 439 kJ mol(−)(1). Even assuming the maximum value (95 % confidence limit) of the observed heat production, glycolysis could account for resynthesis of at most 18 % of the ATP used during the contractions. (ii) When the difference in rates of diffusion of oxygen and heat within the muscle are taken into account, the time courses of oxygen consumption and heat production match each other well during the entire recovery period. The efficiency of recovery (=energy used for ATP synthesis/energy available for ATP synthesis) was estimated from the results. This value, 84.0+/−20.1 % (mean +/− s.e.m., N=29), is relatively high and represents the first such measurement in functioning muscle.

The purpose of the study was to optimise analytical methods for determination of the chemical speciation of mercury in studies of protective mechanisms of selenium. Optimisation of the methods was performed using CRM DOLT-2 (Dogfish liver), both in its original form and after separation of various fractions. The sample was homogenised with 10 mM Tris-HCl buffer (pH 7.6) and ultracentrifuged. The soluble phase obtained was applied to a size exclusion chromatography column (Sephadex ¶G-75 column) for separation of various protein fractions. Total mercury (total Hg), monomethyl mercury (MeHg) and selenium (Se) were determined in whole dogfish liver tissue and its soluble and insoluble phases (pellet). Different approaches for determination of total Hg and MeHg were compared. Simultaneous determination of MeHg and inorganic mercury (Hg2+) was based on alkaline dissolution and/or acid leaching, followed by ethylation, room temperature precollection, isothermal gas chromatography (GC), pyrolysis and detection with cold vapour atomic fluorescence spectrometry (CVAFS). The sum of MeHg and Hg2+ was compared to total Hg results obtained by acid digestion and CVAAS detection. The accuracy of MeHg determination was checked by its determination using acid leaching at room temperature, solvent extraction, back extraction into Milli-Q water, ethylation, GC and CVAFS detection. For the insoluble phase it is recommended to use solvent extraction for MeHg and acid digestion CVAAS for total Hg. For determination of MeHg and Hg2+ in the lyophilised sample and water soluble fractions containing low concentrations of mercury species, the simultaneous measurement of MeHg and Hg2+ after alkaline dissolution is the most appropriate method.

In vivo experiments were conducted on spiny dogfish (Squalus acanthias) and rainbow trout (Oncorhynchus mykiss) in sea water to determine the potential role of externally oriented or gill boundary layer carbonic anhydrase in carbon dioxide excretion. This was accomplished by assessing pH changes in expired water using a stopped-flow apparatus. In dogfish, expired water was in acid–base disequilibrium as indicated by a pronounced acidification (ΔpH=−0.11±0.01; N=22; mean ± S.E.M.) during the period of stopped flow; inspired water, however, was in acid–base equilibrium (ΔpH=−0.002±0.01; N=22). The acid–base disequilibrium in expired water was abolished (ΔpH=−0.005±0.01; N=6) by the addition of bovine carbonic anhydrase (5 mg l−1) to the external medium. Addition of the carbonic anhydrase inhibitor acetazolamide (1 mmol l−1) to the water significantly reduced the magnitude of the pH disequilibrium (from −0.133±0.03 to −0.063±0.02; N=4). However, after correcting for the increased buffering capacity of the water caused by acetazolamide, the acid–base disequilibrium during stopped flow was unaffected by this treatment (control Δ[H+]=99.8±22.8 μmol l−1; acetazolamide Δ[H+]=81.3±21.5 μmol l−1). In rainbow trout, expired water displayed an acid–base disequilibrium (ΔpH=0.09±0.01; N=6) that also was abolished by the application of external carbonic anhydrase (ΔpH=0.02±0.01). The origin of the expired water acid–base disequilibrium was investigated further in dogfish. Intravascular injection of acetazolamide (40 mg kg−1) to inhibit internal carbonic anhydrase activity non-specifically and thus CO2 excretion significantly diminished the extent of the expired water disequilibrium pH after 30 min (from −0.123±0.01 to −0.065±0.01; N=6). Selective inhibition of extracellular carbonic anhydrase activity using a low intravascular dose (1.3 mg kg−1) of the inhibitor benzolamide caused a significant reduction in the acid–base disequilibrium after 5 min (from −0.11±0.01 to −0.07±0.01; N=14). These results demonstrate that the expired water acid–base disequilibrium originates, at least in part, from excretory CO2 and that extracellular carbonic anhydrase in dogfish may have a significant role in carbon dioxide excretion. However, externally oriented carbonic anhydrase (if present in dogfish) plays no role in catalysing the hydration of the excretory CO2 in water flowing over the gills and thus is unlikely to facilitate CO2 excretion.

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.

Recently, the cell-volume-regulated serine-threonine protein kinase h-sgk was cloned from a human hepatoma cell line. The sgk gene was shown to be induced by cell shrinkage in many different mammalian cell lines. In this study, two highly conserved serine-threonine protein kinases, sgk-1 and sgk-2, were cloned from rectal gland tissue of the spiny dogfish (Squalus acanthias). Both kinases showed a distinct pattern of tissue specificity, with high expression levels in kidney, intestine, liver and heart. In rectal gland slices sgk-1 transcription was induced by exposure to hypertonic solution, reduction of the extracellular urea concentration, and addition of the secretagogues vasoactive intestinal polypeptide (VIP) and carbachol. The shark sgk-1 serine-threonine protein kinase may therefore provide a link between cell volume, Cl–secretion and protein phosphorylation state in shark rectal gland cells.

The selectivity of the lipid–protein interactions in trypsinised Na,K-ATPase membranes from Squalus acanthias has been determined by using EPR spectroscopy with different lipid probes spin-labelled on the 14-C atom of the fatty acid chain. From measurements at low ionic strength and different pH values, the pattern of selectivity is: (stearic acid)−>(phosphatidylserine)−>(stearic acid)0>(phosphatidylcholine)±, where superscripts indicate the formal electrostatic charge on the lipid headgroup. This is in the same order as that determined with native Na,K-ATPase membranes [M. Esmann, D. Marsh, Biochemistry 24 (1985) 3572–3578]. The selectivity for phosphatidylserine is independent of pH, over the range pH 6.0–9.0, as found also for native membranes. For membranes trypsinised in the presence of Rb+ ions, and in the presence of Na+ (which allows more extensive proteolysis), the relative association constants, Kr, of all lipids are the same as for control membranes, with the exception of ionised (stearic acid)− that shows the highest specificity. Therefore, both the stoichiometry and the principal determinants of the specificity of lipid–protein interaction are preserved on extensive trypsinisation of Na,K-ATPase membranes. This has implications for the location and arrangement of those amino acid side chains that determine the lipid selectivity of the native Na,K-ATPase.

The ability of the shark antimicrobial aminosterol squalamine to induce the leakage of polar fluorescent dyes from large unilamellar phospholipid vesicles (LUVs) has been measured. Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared from the anionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS), and cardiolipin. Binding analyses based on the leakage data show that squalamine has its highest affinity to phosphatidylglycerol membranes, followed by phosphatidylserine and cardiolipin membranes. Squalamine will also induce the leakage of CF from phosphatidylcholine (PC) LUVs at low phospholipid concentrations. At high phospholipid concentrations, the leakage of CF from PC LUVs deviates from a simple dose–response relationship, and it appears that some of the squalamine can no longer cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather than a generalized disruption of vesicular membranes. By using fluorescently labeled dextrans of different molecular weight, material with molecular weight ≤4000 g/mol is released from vesicles by squalamine, but material with molecular weight ≥10,000 is retained. Negative stain electron microscopy of squalamine-treated LUVs shows that squalamine decreases the average vesicular size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamine/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squalamine solubilizes phospholipid, but only at significantly higher squalamine/phospholipid ratios than required for either dye leakage or vesicle size reduction. Squalamine solubilizes PC at lower squalamine/phospholipid ratios than PG. We suggest that squalamine complexes with phospholipid to form a discrete structure within the bilayers of LUVs, resulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these structures release from the bilayers and aggregate to form either new vesicles or squalamine/phospholipid mixed micelles.

The distribution and nature of natriuretic peptide receptors (NPR) in the gills of dogfish, Squalus acanthias, were examined by tissue section autoradiography, competition analysis, protein electrophoresis, guanylate cyclase (GC) assays, and molecular cloning. Specific NP binding occurred on the gill filaments, but not on the interbranchial septum or gill arch. The binding was densest on the efferent edge of the gills. Higher resolution light-microscopic examination of emulsion-coated sections showed that specific binding occurred mainly on the secondary lamellae and filament body and not on the arterial circulation. At least two types of NPR were revealed. One is linked to GC since NP binding stimulates the production of cGMP. The GC receptor may be similar to the NPR-B mammalian receptor since only pCNP stimulated cGMP production. The second receptor is not linked to GC and binds the specific ligand C-ANF [rat des(Gln 18 , Ser 19 , Gly 20 , Leu 21 , Gly 22 )]. The sequence of a cDNA generated using primers based on conserved regions of vertebrate NPR-C had considerable homology with mammalian and eel NPR-C and eel NPR-D. The presence of GC-linked NPR and NPR-C/NPR-D suggests that the gills are an important target organ for NP action.

Recovery after contraction of white muscle fibres of dogfish was investigated using 31P-NMR and measurements of heat production. The muscle fibres were stimulated to perform either a single isometric tetanus or a series of brief isometric tetani; the NMR measurements showed that approximately half of the phosphocreatine (PCr) was used. The period of activity was followed by a recovery period without stimulation. Both NMR and heat measurements agreed in showing that recovery was very slow, requiring at least 60 min for PCr resynthesis and for the production of recovery heat. The NMR results showed that changes in intracellular pH and in the concentrations of PCr and intracellular phosphate (Pi) had very similar time courses. Intracellular pH moved in the alkaline direction during the period of activity and then returned monotonically during recovery. The non-phosphate buffer power was 13.0±3.1 mmol l−1 intracellular water per pH unit (N=4, mean ± S.E.M.). The results are consistent with the view that oxidative processes resynthesize PCr during recovery, which is slow because of the low mitochondrial content of these muscle fibres.

L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad substrate specificity and utilizes a wide range of 2-oxo acids branched at the C4 atom. Modifications have been made to the sequence of the NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and alter the region of substrate specificity towards various 2-oxocarbonic acids. All variations were based on a 3D-structure model of the enzyme using the X-ray coordinates of the functionally related L-lactate dehydrogenase (L-LDH) from dogfish as a template. This protein displays only 23% sequence identity to L-HicDH. The active site of L-HicDH was modelled by homology to the L-LDH based on the conservation of catalytically essential residues. Substitutions of the active site residues Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify their unique participation in substrate recognition and orientation. The kinetic properties of the L239A, L239M, L236V and T245A enzyme variants confirmed the structural model of the active site of L-HicDH. The substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate, phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the active site of the subsequently refined model. In order to design dehydrogenases with an improved substrate specificity towards keto acids branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236 and Thr245 were introduced and resulted in mutant enzymes with completely different substrate specificities. The substitution T245A resulted in a relative shift of substrate specificity for keto-tert-leucine of more than 17000 compared with the 2-oxocaproate (kcat/KM). For the substrates branched at C4 a relative shift of up to 500 was obtained for several enzyme variants. A total of nine mutations were introduced and the kinetic data for the set of six substrates were determined for each of the resulting mutant enzymes. These were compared with those of the wild-type enzyme and rationalized by the active site model of L-HicDH. An analysis of the enzyme variants provided new insight into the residues involved in substrate binding and residues of importance for the differences between LDHs and HicDH. After the protein design project was complete the X-ray structure of the enzyme was solved in our group. A comparison between the model and the experimental 3D structure proved the quality of the model. All the variants were designed, expressed and tested before the 3D structure became available.

Orthologous homologs of lactate dehydrogenase-A (LDH-A) (EC 1.1.1.27; NAD+:lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent Km) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in Km with increasing measurement temperature occur for all homologs, yet Km at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and Km. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in Km and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in Km between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active sites, and these changes may independently affect kinetic properties and thermal stabilities.

A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome.