Environmental Regulation of Mitochondria-Rich Cells in Chalcalburnus tarichi (Pallas, 1811) During Reproductive Migration

Chalcalburnus tarichi is an anadromous cyprinid fish that has adapted to extreme conditions (salinity 22 ‰, pH 9.8 and alkalinity 153 mEq × l^?1) in Lake Van in eastern Turkey. Changes in immunoreactivity of Na⁺/K⁺-ATPase in gill tissue and osmolarity and ion levels in plasma were investigated in C. tarichi during reproductive migration. Physicochemical characteristics and ion levels in Lake Van were high compared freshwater. Plasma osmolality and plasma ion concentrations ([Na⁺], [K⁺] and [Cl^?]) increased after transfer from freshwater to Lake Van. The mitochondria-rich (MR) cells of the gill were stained in both filament and lamellar epithelia of C. tarichi by immunocytochemistry with a specific antiserum for Na⁺/K⁺-ATPase in river fish samples. Density and area of MR cells were decreased in lake-adapted fishes. These results indicated that freshwater acclimation capacity is correlated with the size and distribution of MR cells in C. tarichi, in contrast to many teleost fishes.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Salinity-dependent modulation by protein kinases and the FXYD2 peptide of gill (Na+, K+)-ATPase activity in the freshwater shrimp Macrobrachium amazonicum (Decapoda,Palaemonidae)

The geographical distribution of aquatic crustaceans is determined by ambient factors like salinity that modulate their biochemistry, physiology, behavior, reproduction, development and growth. We investigated the effects of exogenous pig FXYD2 peptide and endogenous protein kinases A and C on gill (Na⁺, K⁺)-ATPase activity, and characterized enzyme kinetic properties in a freshwater population of Macrobrachium amazonicum in fresh water (<0.5 ‰ salinity) or acclimated to 21 ‰S. Stimulation by FXYD2 peptide and inhibition by endogenous kinase phosphorylation are salinity-dependent. While without effect in shrimps in fresh water, the FXYD2 peptide stimulated activity in salinity-acclimated shrimps by ≈50 %. PKA-mediated phosphorylation inhibited gill (Na⁺, K⁺)-ATPase activity by 85 % in acclimated shrimps while PKC phosphorylation markedly inhibited enzyme activity in freshwater- and salinity-acclimated shrimps. The (Na⁺, K⁺)-ATPase in salinity-acclimated shrimp gills hydrolyzed ATP at a V_max of 54.9 ± 1.8 nmol min^?1 mg^?1 protein, corresponding to ≈60 % that of freshwater shrimps. Mg²⁺ affinity increased with salinity acclimation while K⁺ affinity decreased. (Ca²⁺, Mg²⁺)-ATPase activity increased while V(H⁺)- and Na⁺- or K⁺-stimulated activities decreased on salinity acclimation. The 120-kDa immunoreactive band expressed in salinity-acclimated shrimps suggests nonspecific α-subunit phosphorylation by PKA and/or PKC. These alterations in (Na⁺, K⁺)-ATPase kinetics in salinity-acclimated M. amazonicum may result from regulatory mechanisms mediated by phosphorylation via protein kinases A and C and the FXYD2 peptide rather than through the expression of a different α-subunit isoform. This is the first demonstration of gill (Na⁺, K⁺)-ATPase regulation by protein kinases in freshwater shrimps during salinity challenge.     

Sodium channel activity in brain membrane fractions isolated from rats of different ages

The Na⁺ channel activity (tetrodotoxin sensitive ²²Na⁺ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79–87) from rats 5, 10, 30 and 60 days old. The ²²Na⁺ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 μM) increases the ²²Na⁺ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 μM) causes an increment of the ²²Na⁺ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 μM), the $\text{K}{}_{0.5}$ for veratridine is diminished and the maximum ²²Na⁺ flux is increased. The increments of ²²Na⁺ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin ($\text{K}{}_{0.5}$ approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na⁺ chennel activity. The other subfraction is enriched in myelin and shows no Na⁺ channel actiivty. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na⁺ channel activity.     

Morphofunctional modifications in gill mitochondria-rich cells of Mozambique tilapia transferred from freshwater to 70% seawater, detected by dual observations of whole-mount immunocytochemistry and scanning electron microscopy

Acute responses of gill mitochondria-rich (MR) cells to direct transfer from freshwater to 70% seawater were examined in a euryhaline teleost Mozambique tilapia (Oreochromis mossambicus). Scanning electron microscopic (SEM) observations revealed that apical openings of MR cells were morphologically classified into an apical pit, a convex apical surface, a concave apical surface, and a transitory apical surface. Meanwhile, in whole-mount immunocytochemistry with anti-Na⁺/K⁺-ATPase (NKA), T4 antibody (detecting apical Na⁺/Cl^? cotransporter (NCC) and basolateral Na⁺/K⁺/2Cl^? cotransporter (NKCC)), and anti-Na⁺/H⁺ exchanger-3 (NHE3), NKA-immunoreactive MR cells were functionally classified into immature cells without both NKCC/NCC and NHE3 (type I), ion-absorptive cells with apical NCC (type II), those with apical NHE3 (type III), and ion-secretory cells with basolateral NKCC (type IV). Dual observations of whole-mount immunocytochemistry and SEM clearly showed morphofunctional alterations in MR cells. After transfer to 70% seawater, type-II MR cells with a convex surface or pit closed their apical openings to suspend ion absorption. Type-III MR cells with a concave surface or pit were transformed into type-IV MR cells with an enlarged pit, via a transitory surface. Our findings indicate functional plasticity of type-III/IV MR cells to switch ion-transport functions, whereas type-II MR cells are considered to be specific for freshwater adaptation.     

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda,Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21‰ salinity), as well as potential routes of Na⁺ uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase α-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell invaginations, respectively. These findings are discussed regarding the putative movement of Na⁺ across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations.     

Seaward migrating Atlantic salmon smolts with low levels of gill Na+, K+ -ATPase activity; is sea entry delayed?

Two groups of migrating wild Atlantic salmon (Salmo salar) smolts caught within a 1 week interval in the River Alta, northern Norway, were tagged with acoustic transmitters and measured for gill Na⁺, K⁺ -ATPase activity in order to compare their smolt status with timing of sea entry. The first group of smolts had low levels of gill Na⁺, K⁺ -ATPase activity and resided in the lower part of the river twice as long as the second group that had high levels of gill Na⁺, K⁺ -ATPase activity. This indicates that early migrating smolts may not be completely physiologically adapted for salt water and delay their sea entry, thereby also synchronizing their seaward migration with the later migrating smolts.     

Effects of 5-aminolevulinic acid on Swiss chard (Beta vulgaris L. subsp. cicla) seedling growth under saline conditions

Salinity is a widespread adverse environmental problem globally, and significantly limits crop production. In this study, the possibility of enhancing salinity stress tolerance of Swiss chard (Beta vulgaris L. var. cicla) by 5-aminolevulinic acid (ALA) foliar application was investigated. The Swiss chard plants were grown in hydroponic culture. Twelve-week-old uniform seedlings were treated by 0 and 40 mM saline regimes generated by the mixture of sodium chloride and sodium sulfate (molar ratio NaCl:Na₂SO₄ = 9:1), and were foliar-sprayed with 0 and 60 μM L^?1 ALA (every 3 days) for 6 days; then the plants were treated for another 7 days (every 3 day) with increased concentration of salinity and ALA, 80 mM and 120 μM L^?1. Salinity without ALA application significantly decreased plant growth [43 % in shoot dry weight (DW), 21 % in root DW, 24 % in relative growth rate (RGR), 43 % in leaf area (LA)], water uptake [20.8 % in relative water content (RWC), 47.9 % in osmotic potential (OP)], chlorophyll (Chl) a content (10 %), P_n (36 %), G_s (72 %) and T_r (59 %) compared with those in control plants; however, under saline conditions, ALA foliar application improved plant growth (49.7 % in shoot DW, 27 % in root DW, 42.3 % in RGR, 72.1 % in LA) and increased RWC (12 %), Chl a content (10 %) and photosynthetic parameters (27 % in P_n, 28 % in G_s, 14 % in T_r) compared with those in untreated plants. Salinity significantly increased Na⁺ content, resulting in the reduction of Mg²⁺ and K⁺ contents. ALA foliar application alleviated ionic toxicity through the reduction of Na⁺ content and Na⁺/K⁺ ratio. On the other hand, it increased total nitrogen and glycine betaine (GB) content. ALA foliar application slightly reduced malondialdehyde (MDA) content, indicating that ALA has the potential to alleviate oxidative stress in salinity-stressed Swiss chard.     

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA–dT)]2, [poly(dG–dC)]2, and calf thymus DNA

The interaction of the Cu(II) drugs CuL(NO₃) and CuL′(NO₃) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL′ is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL]⁺ and [CuL′]⁺) with [poly(dA–dT)]₂, [poly(dG–dC)]₂, and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 °C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA–dT)]₂, with a site size n = 6–7, whereas they act as external binders with [poly(dG–dC)]₂ and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL′]⁺ were slightly larger than with [CuL]⁺. The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL]⁺ and [CuL′]⁺, with a significative dose–response effect for [CuL′]⁺, which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH₂ group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.     

The effects of gill remodeling on transepithelial sodium fluxes and the distribution of presumptive sodium-transporting ionocytes in goldfish (Carassius auratus)

Goldfish, Carassius auratus, adaptively remodel their gills in response to changes in ambient oxygen and temperature, altering the functional lamellar surface area to balance the opposing requirements for respiration and osmoregulation. In this study, the effects of thermal- and hypoxia-mediated gill remodeling on branchial Na⁺ fluxes and the distribution of putative Na⁺-transporting ionocytes in goldfish were assessed. When assessed either in vitro (isolated gill arches) or in vivo at a common water temperature, the presence of an interlamellar cell mass (ILCM) in fish acclimated to 7°C clearly decreased Na⁺ efflux across the gill relative to fish maintained at 25°C and lacking an ILCM. However, loss of the ILCM in 7°C-acclimated fish exposed to hypoxia led to a decrease in Na⁺ efflux (assessed under hypoxic conditions) despite the apparent large increases in functional lamellar surface area. Goldfish possessing an ILCM were able to sustain Na⁺ uptake, albeit at a lower rate matched to efflux, owing to the re-distribution of ionocytes expressing genes thought to be involved in Na⁺ uptake [Na⁺/H⁺ exchanger isoform 3 (NHE3) and V- type H⁺-ATPase] to the edge of the ILCM where they can establish contact with the surrounding environment. NHE-expressing cells co-localized with Na⁺/K⁺-ATPase expression, suggesting a role for NHE in Na⁺-uptake in the goldfish. Implications of the ILCM on ion fluxes in the goldfish are discussed.     

Mechanisms of Na+ uptake,ammonia excretion,and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi,Hemigrammus rhodostomus,and Moenkhausia diktyota)

Mechanisms of Na⁺ uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J _in), efflux (J _out), and net flux rates (J _net) of Na⁺ and Cl^?, and the net excretion rate of ammonia (J _Amm). The fish were collected directly from the Rio Negro, and studied in their native “blackwater” which is acidic (pH 4.5), ion-poor (Na⁺, Cl^? ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l^?1). J _in ^Na , J _in ^Cl , and J _Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J _in ^Na was unaffected by amiloride (10^?4 M, NHE and Na⁺ channel blocker), but both J _in ^Na and J _in ^Cl were virtually eliminated (85–99 % blockade) by AgNO₃ (10^?7 M). A time course study on cardinal tetras demonstrated that J _in ^Na blockade by AgNO₃ was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10^?4 M) caused a 50 % reduction in J _in ^Na _.. Additionally, J _in ^Na was unaffected by phenamil (10^?5 M, Na⁺ channel blocker), bumetanide (10^?4 M, NKCC blocker), hydrochlorothiazide (5 × 10^?3 M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J _in ^Na (by acetazolamide and AgNO₃ respectively), had any inhibitory effect on J _Amm. Therefore, Na⁺ uptake in Rio Negro tetras depends on an internal supply of H⁺ from CA, but does not fit any of the currently accepted H⁺-dependent models (NHE, Na⁺ channel/V-type H⁺-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current “Na⁺/NH₄ ⁺ exchange metabolon” paradigm. Na⁺, K⁺-ATPase and V-type H⁺-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH₄Cl, 10^?3 M) significantly increased J _in ^Na , and NH₄ ⁺ was equally or more effective than K⁺ in activating branchial Na⁺,(K⁺) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na⁺ uptake, but that Na⁺ uptake (by an as yet unknown H⁺-dependent apical mechanism) depends on ammonia excretion, driven by active NH₄ ⁺ entry via basolateral Na⁺,(K⁺)-ATPase.     

Effects of Na+ and/or K+ on the Mg2+-dependent ATPase activities in shrimp (Macrobrachium amazonicum) gill homogenates

• 1.1. Homogenates of gills from the freshwater shrimp M. amazonicum exhibit the following ATPase activities: (i) a basal, Mg²⁺-dependent ATPase; (ii) an ouabain-sensitive, Na⁺ + K⁺-stimulated ATPase; (iii) an ouabain-insensitive, Na⁺-stimulated ATPase; and (iv) an ouabain-insensitive, K⁺-stimulated ATPase.
• 2.2. K⁺ suppresses the Na⁺-stimulated ATPase activity in a mixed-type kind of inhibition, whereas Na⁺ does not exert any noticeable effect on the K⁺-stimulated ATPase activity.
• 3.3. The Na⁺- and the K⁺-stimulated ATPase activities are totally inhibited by 5 mM ethacrynic acid in the incubation medium.
• 4.4. The Na⁺- and the K⁺-stimulated ATPase activities are not expressions of the activation of a Ca-ATPase.
• 5.5. The possible localization and roles of the described ATPases within the gill epithelium are briefly discussed and evaluated.

     

Physiological and proteomic analysis of salinity tolerance of the halotolerant cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp

The halotolerant cyanobacterium Anabaena sp was grown under NaCl concentration of 0, 170 and 515 mM and physiological and proteomic analysis was performed. At 515 mM NaCl the cyanobacterium showed reduced photosynthetic activities and significant increase in soluble sugar content, proline and SOD activity. On the other hand Anabaena sp grown at 170 mM NaCl showed optimal growth, photosynthetic activities and comparatively low soluble sugar content, proline accumulation and SOD activity. The intracellular Na⁺ content of the cells increased both at 170 and 515 mM NaCl. In contrast, the K⁺ content of the cyanobacterium Anabaena sp remained stable in response to growth at identical concentration of NaCl. While cells grown at 170 mM NaCl showed highest intracellular K⁺/Na⁺ ratio, salinity level of 515 mM NaCl resulted in reduced ratio of K⁺/Na⁺. Proteomic analysis revealed 50 salt-responsive proteins in the cyanobacterium Anabaena sp under salt treatment compared with control. Ten protein spots were subjected to MALDI-TOF–MS/MS analysis and the identified proteins are involved in photosynthesis, protein folding, cell organization and energy metabolism. Differential expression of proteins related to photosynthesis, energy metabolism was observed in Anabaena sp grown at 170 mM NaCl. At 170 mM NaCl increased expression of photosynthesis related proteins and effective osmotic adjustment through increased antioxidant enzymes and modulation of intracellular ions contributed to better salinity tolerance and optimal growth. On the contrary, increased intracellular Na⁺ content coupled with down regulation of photosynthetic and energy related proteins resulted in reduced growth at 515 mM NaCl. Therefore reduced growth at 515 mM NaCl could be due to accumulation of Na⁺ ions and requirement to maintain higher organic osmolytes and antioxidants which is energy intensive. The results thus show that the basis of salt tolerance is different when the halotolerant cyanobacterium Anabaena sp is grown under low and high salinity levels.     

Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology

Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70–80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r ²?=?0.946–0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.     

The copper-molybdenum antagonism in ruminants. III. Reaction of copper(II) with tetrathiomolybdate(VI)

The stoichiometry of the reaction between Cu(II) and MoS₄²⁻ in neutral aqueous solution was observed to proceed with a 1.5:1 Cu:Mo ratio. The reaction results in the reduction of Cu(II) and the quantitative formation of an insoluble solid. The results contrast with an earlier report of a 1:1 stoichiometric ratio, this latter ratio was, however, observed with Cu^II(albumin) as reactant, in this case no precipitate was observed. The insoluble products were examined by a number of spectroscopic techniques and by X-ray power diffraction and elemental analysis. Two products were identified. Solid A is isostructural with the known (NH₄)Cu^IMoV^VIS₄, i.e. has the composition M^ICuMoS₄, M^I= NH₄⁺, Na⁺ or Et₄N⁺. Solid B has the approximate composition CuMoS₄O_x, x=2−3 and contains Cu(I) and Mo(V) centres. Formation of compound B therefore involves an unusual internal two-electron redox process. The reaction and products are of particular biological significance.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

The effect of Cu on gill and esophagus lipid metabolism in the rainbow trout Oncorhynchus mykiss

Rainbow trout Oncorhynchus mykiss were exposed to 0, 100, 300 and 800 μgl⁻¹ ambient Cu in brackish water (BW) for 4 days at 13 °C and subsequently transferred to either clean BW, clean fresh water (FW) or clean seawater (SW) at 16 °C. After incubation with ³²P-phosphate and ¹⁴C-acetate added as precursors to the water the fish showed a degradation, depending on previous [CU], of ³²P- and ¹⁴C-labelled gill membrane phospholipids if they had been transferred to SW or remained in BW. Corresponding experiments where the fish were exposed to Cu in BW for 12 days showed a similar subsequent degradation in SW and BW of both gill and esophagus membrane phospholipids, however to a much lesser degree in gill tissue than after 4 days. Plasma Na⁺ was similarly reduced by up to 8%, depending on previous ambient Cu, but in this case only after transfer to FW and only after 4 days of exposure. Both the effect on membrane lipid metabolism and plasma Na⁺ thus showed acclimation to ambient Cu but there was apparently no direct correlation between the two different types of observed changes in membrane function.