Comparison of the binding properties of A1 adenosine receptors in brain membranes of two congeneric marine fishes living at different depths

Summary The binding properties of A₁ adenosine receptors in brain membranes were compared in two congeneric marine teleost fishes which differ in their depths of distribution. Adenosine receptors were labeled using the A₁ selective radioligand [³H]cyclohexyladenosine ([³H]CHA). The A₁ receptor agonist [³H]CHA bound saturably, reversibly and with high affinity to brain membranes prepared fromSebastolobus altivelis andS. alascanus; however, the meanK _d values differed significantly (Figs. 1–3, Table 1). Saturation data fit to a one site model indicated that the A₁ receptor inS. alascanus exhibited a higher affinity (K _d=1.49 nM) for [³H]CHA whereas A₁ receptors inS. altivelis exhibited a significantly lower affinity (K _d=3.1 nM). Moreover,S. altivelis, but notS. alascanus, parameter estimates for [³H]CHA binding to two sites of receptor were obtained (Fig. 3, Table 1). The mean dissociation constant values for the high and low affinity sites for [³H]CHA inS. altivelis were 0.43 nM and 16.3 nM, respectively. In equilibrium competition experiments the adenosine analogs R-phenylisopropyladenosine (R-PIA), N-ethylcarboxamidoadenosine (NECA) and S-phenylisopropyladenosine (S-PIA) all displayed higher affinities for A₁ receptors inS. alascanus as compared toS. altivelis brain membranes (Table 2, Fig. 6). The specific binding of [³H]CHA was significantly increased by 0.1 and 1.0 mM MgCl₂ in both fishes; however, the sensitivity (95–131% increase) ofS. altivelis to this effect was significantly greater than that ofS. alascanus (48–91% increase) (Fig. 5). The results of kinetic, equilibrium saturation and equilibrium competition experiments all suggest that A₁ adenosine receptors ofS. altivelis andS. alascanus brain membranes differ with respect to their affinities for selected adenosine agonists.Abbreviations CHA cyclohexyladenosine - R-PIA R-phenylisopropyladenosine - S-PIA S-phenylisopropyladenosine - NECA N-ethylcarboxamidoadenosine - NEM N-ethylmaleimide - 2-ClAdo 2-chloroadenosine - GTP guanosine triphosphate - N protein guanine nucleotide binding protein - _{n H} Hill slope     

Pressure-adaptive differences in proteolytic inactivation of M4-lactate dehydrogenase homologues from marine fishes

The inactivation by hydrostatic pressure of muscle-type lactate dehydrogenase (M4-LDH, EC 1.1.1.27; L-lactate: NAD+ oxidoreductase) homologues from five shallow-living and six deep-living marine teleost fishes was compared. The pressures which inactivate these enzymes are much higher than the pressures experienced by any of the species. To determine whether hydrostatic pressure effects on protein aggregation state and conformation might influence proteolysis, the inactivation of LDH by the proteases, trypsin (EC 3.4.21.4) and subtilisin (EC 3.4.4.16) was determined at atmospheric pressure and 1,000 atm pressure. At 10 degrees C and atmospheric pressure, the enzymes of the shallow-living fishes are inactivated four times faster by trypsin and three times faster by subtilisin than are the homologues of the deep-living species. At 1,000 atm pressure, the homologues of shallow-occurring fishes were inactivated 28 to 64% more than predicted from the summed effects of denaturation by 1,000 atm pressure and tryptic inactivation at atmospheric pressure. In contrast, the homologues of the deep-sea species were inactivated by trypsin 0 to 21% more than expected. At 1,000 atm, inactivation by subtilisin increased to a similar degree for enzymes from both deep- and shallow-living species. However, at 1,000 atm, the M4-LDH homologues of the deep-sea species lost less activity (55.3%) than did the homologues of the shallow species (86.4%). In comparisons made at 200 atm, a pressure typical of the habitat of the deep-occurring species, tryptic inactivation of the LDH of the shallow-living Sebastes melanops was increased 14%. No pressure inactivation of the enzyme is evident at 200 atm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis at pressure and HPLC peptide mapping of M4-lactate dehydrogenase homologs from marine fishes living at different depths.

1. The effects at 10 degrees C of moderate hydrostatic pressure (136 atm) on trypsinolysis of muscle-type (M4) lactate dehydrogenase homologs (LDH, EC 1.1.1.27, L-lactate:NAD+ oxidoreductase) from shallow- and deep-occurring marine fishes were examined by mapping the partial digests by reverse phase HPLC. 2. Comparison of peptide maps of digests generated at 1 and 136 atm revealed that increased pressure did not expose new cleavage sites in homologs of any of the species; no new peptides were generated. 3. Increased pressure did alter the relative amounts of peptides produced. The net effect of increased pressure was to increase the amount of peptides generated in the shallow-occurring species. For deep-living species pressure did not alter the net amount of peptides produced compared to the 15 min atmospheric pressure samples, although the relative amounts of some of the peptides changed. Incubation at 136 atm for 30 min decreased the net amount of peptides produced. 4. It is suggested that the effects of pressure on trypsinolysis may result from slight conformational changes in the substrate proteins.     

Pressure-adaptive differences in the binding and catalytic properties of muscle-type (M4) lactate dehydrogenases of shallow- and deep-living marine fishes

Summary The pressure sensitivities of substrate (pyruvate) and cofactor (NADH) binding and catalytic rate of purified muscle-type (M₄) lactate dehydrogenases (LDH, EC 1.1.1.27; NAD⁺: lactate oxidoreductase) from shallow- and deep-living teleost fishes were compared. The LDH's of the shallow species are significantly more pressure-sensitive than the LDH's of the deep-living fishes. The apparent Michaelis constant (K _m)¹ of pyruvate of the deep-living species' LDH's is pressure-insensitive over the entire pressure range used in these studies, 1 to 476 atmospheres (Fig. 1). For the LDH's of the shallow species, theK _m of pyruvate increases significantly between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres. TheK _m of NADH displays a much higher pressure sensitivity. For the LDH's of the deep species, theK _m of NADH increases slightly (approximately 32%) between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres (Fig. 1). TheK _m of the shallow species' LDH's rises sharply (approximately 113%) between 1 and 68 atmospheres, and then continues to increase at a slower rate up to 476 atmospheres. This marked inhibition of cofactor binding by pressure for the shallow species' LDH's may be of sufficient magnitude to seriously impair the function of these LDH's at pressures typical of those encountered by the deeper-living species.Pressure effects on optimal velocity, measured under high (optimal) concentrations of pyruvate and NADH, were generally lower for the LDH's of the deep species (Table 1).These results indicate that M₄-LDH's of shallow water fishes are not pre-adapted for function at deepsea pressures, and that the reduction of pressure sensitivities ofK _m's and catalysis may be a ubiquitous feature of adaptation to life at depth. The virtually identical pressure responses of M₄-LDH's from deepliving teleosts belonging to four different families represents a striking example of convergent evolution at the molecular level.     

Temperature-adaptive differences in the kinetic properties of muscle-type lactate dehydrogenases of congeneric rocky-shore fishes from the Sea of Cortez

Four species of groupers, genus Epinephelus, exhibiting overlapping distribution ranges in the middle and lower regions of the Sea of Cortez, were studied in terms of biochemical, genetic, and functional enzymic features. The results of enzymatic assays of muscle-type lactate dehydrogenases (M₄-LDH) from tropical-, subtropical- and temperate-zone groupers showed thermal compensatory differences in their kinetic properties (apparent K_m of pyruvate and catalytic rate constant, k_cat). These kinetic adaptations are reflected in a strong conservation of the functional characteristics of the enzyme at the mean temperature of their habitats which differ by 3–6°C on the average. These results point out that minor differences in habitat (body) temperature are sufficient to favor the evolution of functional adaptations in the enzymes of the species. The use of closely related congeneric species inhabiting different thermal environments is a valuable approach to the study of molecular evolution at a fine scale level.     

Urea-requiring lactate dehydrogenases of marine elasmobranch fishes

Summary The kinetic properties — apparentK _m of pyruvate, pyruvate inhibition pattern, and maximal velocity — of M₄ (skeletal muscle) lactate dehydrogenases of marine elasmobranch fishes resemble those of the homologous lactate dehydrogenases of non-elasmobranchs only when physiological concentrations of urea (approximately 400 mM) are present in the assay medium. Urea increases the apparentK _m of pyruvate to values typical of other vertebrates (Fig. 2), and reduces pyruvate inhibition to levels seen with other M₄-lactate dehydrogenases (Fig. 3). Urea reduces the activation enthalpy of the reaction, and increasesV _max at physiological temperatures (Fig. 4).The M₄-lactate dehydrogenase of the freshwater elasmobranch,Potamotrygon sp., resembles a teleost lactate dehydrogenase, i.e., although it is sensitive to urea, it does not require the presence of urea for the establishment of optimal kinetic properties.     

Life in the unthinking depths: energetic constraints on encephalization in marine fishes

Several hypotheses have been proposed to explain the limitation of brain size in vertebrates. Here, we test three hypotheses of brain size evolution using marine teleost fishes: the direct metabolic constraints hypothesis (DMCH), the expensive tissue hypothesis and the temperature‐dependent hypothesis. Our analyses indicate that there is a robust positive correlation between encephalization and basal metabolic rate (BMR) that spans the full range of depths occupied by teleosts from the epipelagic (< 200 m), mesopelagic (200–1000 m) and bathypelagic (> 4000 m). Our results disentangle the effects of temperature and metabolic rate on teleost brain size evolution, supporting the DMCH. Our results agree with previous findings that teleost brain size decreases with depth; however, we also recover a negative correlation between trophic level and encephalization within the mesopelagic zone, a result that runs counter to the expectations of the expensive tissue hypothesis. We hypothesize that mesopelagic fishes at lower trophic levels may be investing more in neural tissue related to the detection of small prey items in a low‐light environment. We recommend that comparative encephalization studies control for BMR in addition to controlling for body size and phylogeny.     

Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases

Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in 2H2O. When the reactions catalyzed by alanine racemase (EC 5.1.1.1) and L-alanine dehydrogenase (EC 1.4.1.1), which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in 2H2O, [4R-2H]NADH is exclusively produced. Similarly, [4S-2H]NADH is made in 2H2O with amino-acid racemase with low substrate specificity (EC 5.1.1.10) and L-leucine dehydrogenase (EC 1.4.1.9), which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by 1H NMR measurement without isolation of the coenzymes and products.     

Characterization of NAD-dependent malate dehydrogenases from spinach leaves

Summary. Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different K _m values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools. Correspondence: T. Cvetić, Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia.     

抽穗期不同灌水深度下水稻群体与大气的温度差异

于2005年选用3个水稻品种(扬稻6号、扬粳9538和武香粳14)在抽穗开花期设置无水(0 cm)、浅水(2～4 cm)和深水(10 cm以上)3个水分处理,研究水分管理对水稻不同部位温度的影响.结果表明,在10:30-15:00,田间灌水深度显著影响水稻植株不同部位的温度.田间水位越高,水稻各部位的温度越低,大气温度与植株不同部位的温度的差值越高.深水管理下,3个品种大气与水稻穗部、植株中部和田面温差的平均值分别比无水管理高1.37、2.98和4.12 ℃,比浅水管理高0.67、1.59和2.17 ℃;而浅水管理大气与下穗部、植株中部和田面温差的平均值分别比无水管理高0.71、1.39和1.95 ℃.不同品种大气与水稻各部位的温度差值也存在差异.对田面-植株中部-穗部温度传递特性的分析表明,不同水深管理下的水稻群体内部温度的变化梯度符合热学传递原理,说明在抽穗期高温来临之前提高田间水深对降低或缓解水稻高温热害具有明显作用.     

Incidence and severity of marine borer attack at different depths at Mtongwe Jetty Pontoon Mombasa,Kenya

The activity of marine borers was investigated at Mtongwe Jetty Pontoon, Mombasa, Kilindini harbour using untreated panels of Pinus patula (Schlecht) submerged to different depths. Twenty test panels replicated over 7 months were each strung in three ladder‐like frames using nylon twine and suspended vertically, in such a way that the top most panels were 25 cm below the water surface at low and high tide. Monthly observations were made between January and July for the vertical incidence and extent of marine borer attack following the technique of Bobat (1995) . Marine borers were collected from test panels, identified and counted. In addition, the percentage weight loss for panels at different depths was determined at the end of 7 months. Teredo fulleri (Clapp) was dominant on panels near water surface while Martesia striata (Linne) and Lyrodus pedicellatus (Quatrefages) were predominant at the bottom. The results showed that attack began within the first month of exposure and was severest by the seventh month. The vertical incidence and extent of attack decreased with increasing depth of panel exposure and was negatively correlated with depth. This is attributed to changes in hydrographic conditions.     

The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low K _m and one with a high K _m for GTP. The high-affinity GTPase activity, characteristic of the subunits of the guanine nucleotide binding protein pool, was stimulated by the A₁ adenosine receptor agonists N ⁶(R-phenylisopropyl)adenosine and N ⁶-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N ⁶ (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 mol·1^-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent K _m of GTP is decreased approximately 10% in each of the species, and the V _max values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased K _m of GTP and the increased V _max ranged from-7.0 to-9.9 ml·mol^-1. Increased pressure markedly decreased the efficacy of N ⁶ (R-phenylisopropyl) adenosine, N ⁶-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A₁ adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.Abbreviations [³H] DPCPX ³H cyclopentyl-1, 3-dipropylxanthine - AppNHp 5-adenylylimidodiphosphate - cpm counts per minute - CPA N ⁶-cyclopentyladenosine - EDTA ethylenediaminetetra acetic acid - EGTA ethyleneglycol-bis (-aminoethylether) N, N, N, N-totra-acctic acid - G protein guanine nucleotide binding protein - G_i inhibitory G protein - G_o other G protein, common in brain membranes - G_s stimulatory G protein - GTPase guanosine triphosphatase - K _i inhibition constant - K _m Michaelis constant - pK _a log of the dissociation constant - R-PIA N ⁶ (R-phenylisopropyl) adenosine - TRIS tris[hydroxymethyl]aminomethane - V_max maximal velocity - [-³²P]GTP [-³²P] guanosine 5-triphosphate (tetra (triethylammonium) salt)     

Size at maturity of Mediterranean marine fishes

In this review we collected data on the length at maturity (L_m) and maximum reported total length (L_max) of 565 Mediterranean marine fish stocks, representing 150 species, 68 families, 24 orders and 3 classes. Overall, L_m ranged from 2 cm, for the males of the toothcarp Aphanius fasciatus, to 350 cm, for the females of the bluntnose sixgill shark Hexanchus griseus. L_m was positively linearly related with L_max for Actinopterygii (logL_m = ?0.123 + 0.92 × logL_max; r ² = 0.87, n = 471, P < 0.001) and Elasmobranchii (logL_m = ?0.008 + 0.922 × logL_max; r ² = 0.90, n = 92, P < 0.001) with the two slopes being significantly different (ANCOVA: F = 2,904, P < 0.001). The reproductive load (L_m/L_max) ranged between 0.23 (sand steenbras Lithognathus mormyrus) and 0.94 (angular roughshark Oxynotus centrina and thornback ray Raja clavata). The mean L_m/L_max was significantly (ANOVA, F = 34.14, P < 0.001) lower for Actinopterygii (mean = 0.59, SD = 0.122, n = 471) compared to Elasmobranchii (mean = 0.70, SD = 0.132, n = 92) and Holocephali (mean = 0.77, SD = 0.077, n = 2). The L_m/L_max was significantly (ANOVA, F = 43.80, P < 0.001) higher for species providing some form of parental care, i.e. guarders, bearers, nesters (mean L_m/L_max ± SD = 0.68 ± 0.141, n = 111) compared to non-guarders (mean L_m/L_max ± SD = 0.59 ± 0.123, n = 454). The mean L_m/L_max displayed a remarkable constancy with longitude (northern and southern Mediterranean coastline: ANOVA, F = 0.01, P = 0.93), latitude (western, central and eastern regions: ANOVA, F = 1.25, P = 0.29) and habitat (ANOVA, F = 0.85, P = 0.51).     

Comparative behaviour of two species of Lepadogaster (Pisces: Gobiesocidae) living at different depths

The two clingfish species studied occupied similar habitats but occurred at different depths. When compared with the subtidal species Lepadogaster candollei , the intertidal species Lepadogaster l. purpurea was less active, spent more time in shelters, visited fewer shelters, showed more site fidelity, and spent less time swimming. Feeding, swimming, and agonistic behaviours were performed mainly in close contact with the substrate in this species. It is hypothesized that these contrasts in behaviour may have evolved under different levels of turbulence.     

A new enzyme that specifically inactivates apo-protein of NAD-dependent dehydrogenases

The existence of a new inactivating enzyme for NAD-dependent enzymes was recognized in small intestine of niacin-deficient rats. The increase of the enzyme activity was in parallel with the degree of niacin deficiency. The enzyme splits apo-NAD dependent enzymes to smaller molecular compounds. The inactivating reaction is prevented by the presence of NAD.     

Modelling patterns of coexistence of three congeneric headwater fishes

1. Mechanisms driving patterns of occurrence and co-occurrence among North American freshwater fishes are poorly understood. In particular, the influence of biotic interactions on coexistence among stream reaches and their effects on regional species distribution patterns is not well understood for congeneric headwater fishes.
2. Occupancy models provide a useful framework for examining patterns of co-occurrence while also accounting for imperfect detection. Occupancy models may be extended to test for evidence that a dominant species influences the occurrence of a subordinate species and thus evaluate support for the hypothesis that species interactions drive patterns of coexistence.
3. We examined patterns of occurrence and co-occurrence at the stream-reach scale among three species of darters (Percidae: Etheostomatinae) that occupy headwater streams within a Gulf Coastal Plain drainage in the south-eastern U.S.A. We assessed species occurrences at 97 sites in first- to third-order streams on one occasion each and used data from four sub-reaches sampled with equal effort at each site to estimate species-specific detection probabilities. Following sampling, a suite of habitat variables was collected at three equidistant points along each of the three transects established within a sub-reach. Coarse (stream-segment, catchment, network) scale variables were also incorporated using geospatial data. Single-species and two-species occupancy models were used to examine patterns of occupancy and coexistence.
4. The occupancy of each species was influenced by distinct habitat variables. Goldstripe darters (Etheostoma parvipinne) were constrained by a stream size gradient, groundwater input appeared to influence the occurrence of Yazoo darters (Etheostoma raneyi), and local habitat heterogeneity (e.g. variation in depth and current velocity) appeared to influence the occupancy of redspot darters (Etheostoma artesiae).
5. We found no evidence that the presence of one species influenced the occurrence of another within a stream-reach based on two-species occupancy models. Rather, species co-occurrences were best explained as independent occurrences within a stream-reach according to species-specific habitat associations.
6. Occupancy modelling may provide a suitable framework for evaluating the influence of biotic interactions among congeneric stream fishes along species-specific habitat gradients at the stream reach scale. Our study offers insight into how habitat variation can influence coexistence of potential competitors across a large river system.

     

Occurrence ofSagittaria sagittifolia at different depths of water

The authors observations onSagittaria sagittifolia L. at 100 natural localities were compared with data in the literature. The habitat of the species is between 0 and 0.8 m of water depth; flowering and fruiting are optimal between 0.1 and 0.8 m.