Effects of iso-osmotic NaCl and mannitol on growth, proline content, and antioxidant defense in Mammillaria gracilis Pfeiff. in vitro-grown cultures

Effects of iso-osmotic concentrations of NaCl and mannitol were studied in Mammilaria gracilis (Cactaceae) in both calli and tumors grown in vitro. In both tissues, relative growth rates were reduced under osmotic stress, which were accompanied by a decrease in both tissue water and K⁺ content. However, growth was inhibited to a lesser extent after exposure to NaCl, when accumulation of Na⁺ ions was observed. In calli, only salinity increased proline content, whereas with tumors proline accumulated after both osmotic stresses. Osmotic stresses also induced oxidative damage in both cactus tissues, although higher oxidative injury was caused by mannitol in calli and by salt in tumors. Low iso-osmotic concentrations of NaCl (75 mM) and mannitol (150 mM) increased peroxidase, ascorbate peroxidase, and esterase activities, whereas elevated catalase activity was recorded only after mannitol treatment in both tissues. High osmotic stress generally decreased enzymatic activities. However, in calli, esterase activity increased in response to high salinity, whereas ascorbate peroxidase activity was enhanced after high mannitol stress. In conclusion, both in vitro-grown cactus tissues were found to be sensitive to osmotic stress caused by either mannitol or NaCl, but accumulation of Na⁺ ions in response to salt somewhat contributed to osmotic adjustment. However, more prominent oxidative damage induced by NaCl compared to mannitol in tumor could be related to ion toxicity. The mechanisms that mediate responses to salt- and mannitol-induced osmotic stresses differed and were dependent on tissue type.     

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na⁺, K⁺-ATPase, H⁺-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na⁺, K⁺-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na⁺. Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na⁺ import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.     

Is osmotic potential a more appropriate property than electrical conductivity for evaluating whole-plant response to salinity?

Studies of whole-plant or crop responses to salinity often focus on yield or growth reduction in terms of solution ion concentration or electrical conductivity. The response functions describing salt stress may be better presented in terms of solution osmotic potential. We looked at the effect of increasing concentrations of NaCl and CaCl₂, either alone or in equinormal combination, on three different plant species: bean (Phaseolus vulgaris L.), corn (Zea mays L.) and melon (Cucumis melo L.). Corn and melon were found to be relatively tolerant and beans more sensitive to salinity. When yield response was related to the electrical charge concentration of the salts, i.e. salinity was expressed in units of mequiv. L^?1 or electrical conductivity, the stress effects of Na and Ca appeared to be of different magnitudes: plant growth was more sensitive to excess Na than to excess Ca and the effect of combined Na and Ca was intermediate. The effects of the two salts were, however, indistinguishable when salinity was expressed in terms of osmotic potential of the water. For all three species, the response curves of yield as a function of level of equipotential solutions of NaCl, CaCl₂ or combinations of the two salts practically overlapped. Presentation and interpretation of the whole-plant salinity response in terms of osmotic potential would be beneficial in attempts to differentiate between the osmotic and toxic effects of salinity, in normalizing data sets and in increasing their relevance in practical applications.     

Salt tolerance strategies of Lygeum spartum L.: A new fodder crop for Algerian saline steppes

Lygeum spartum L. is a native species in Algerian salt steppes. The plant is of interest because of its tolerance to environmental stresses and its use as a fodder grass for livestock in low-rainfall Mediterranean areas. Nevertheless, plant responses of this plant to salt stress are still not investigated in detail. Therefore, L. spartum L. was grown in hydroponic conditions to investigate the effect of salinity (0, 30, 60 and 90 mM NaCl) on growth, water relations, gas exchange, leaf chlorophyll concentration, glycine betaine and mineral uptake. Plant growth was reduced at 60 and 90 mM NaCl, but was not significantly lower than in the controls at 30 mM NaCl. Sodium (Na⁺), chloride (Cl⁻) and glycine betaine contents in plants increased, whereas calcium (Ca²⁺), potassium (K⁺), relative water content (RWC), root hydraulic conductivity (L₀) and chlorophyll content decreased with an increase in salinity. Water potential (Ψ_ω) and osmotic potential (Ψ_π) of plants decreased with an increase in salinity. No change was observed in the turgor potential (Ψ_τ). Photosynthesis parameters (CO₂ assimilation rate, stomatal conductance and transpiration rate) did not change significantly at 30 mM NaCl, as compared to the control. Higher salt levels impaired photosynthetic capacity of L. spartum mainly via a stomatal limitation leading to a low CO₂ assimilation rate. This might be a consequence of the reduced whole-plant hydraulic conductivity under salt stress. The results demonstrated that L. spartum L. can be characterised as a moderately salt-tolerant species. Salt tolerance in this species is achieved by appropriate osmotic adjustment involving accumulation of ions and glycine betaine. At high salinities, growth reduction probably occurs as a result of high concentrations of Na⁺ and Cl⁻ and their interference with other ions such as Ca²⁺ and K⁺. This plant can be used locally as a fodder for livestock and to stabilise sand dunes and rehabilitate salt soils.     

The Comparative Osmoregulatory Ability of Two Water Beetle Genera Whose Species Span the Fresh-Hypersaline Gradient in Inland Waters (Coleoptera: Dytiscidae,Hydrophilidae)

A better knowledge of the physiological basis of salinity tolerance is essential to understanding the ecology and evolutionary history of organisms that have colonized inland saline waters. Coleoptera are amongst the most diverse macroinvertebrates in inland waters, including saline habitats; however, the osmoregulatory strategies they employ to deal with osmotic stress remain unexplored. Survival and haemolymph osmotic concentration at different salinities were examined in adults of eight aquatic beetle species which inhabit different parts of the fresh—hypersaline gradient. Studied species belong to two unrelated genera which have invaded saline waters independently from freshwater ancestors; Nebrioporus (Dytiscidae) and Enochrus (Hydrophilidae). Their osmoregulatory strategy (osmoconformity or osmoregulation) was identified and osmotic capacity (the osmotic gradient between the animal’s haemolymph and the external medium) was compared between species pairs co-habiting similar salinities in nature. We show that osmoregulatory capacity, rather than osmoconformity, has evolved independently in these different lineages. All species hyperegulated their haemolymph osmotic concentration in diluted waters; those living in fresh or low-salinity waters were unable to hyporegulate and survive in hyperosmotic media (> 340 mosmol kg^-1). In contrast, the species which inhabit the hypo-hypersaline habitats were effective hyporegulators, maintaining their haemolymph osmolality within narrow limits (ca. 300 mosmol kg^-1) across a wide range of external concentrations. The hypersaline species N. ceresyi and E. jesusarribasi tolerated conductivities up to 140 and 180 mS cm^-1, respectively, and maintained osmotic gradients over 3500 mosmol kg^-1, comparable to those of the most effective insect osmoregulators known to date. Syntopic species of both genera showed similar osmotic capacities and in general, osmotic responses correlated well with upper salinity levels occupied by individual species in nature. Therefore, osmoregulatory capacity may mediate habitat segregation amongst congeners across the salinity gradient.     

Proline Accumulation in Transgenic Tobacco as a Result of Expression of Arabidopsis Δ1-Pyrroline-5-carboxylate synthetase (P5CS) During Osmotic Stress

The entire coding sequence of the bi-functional enzyme, Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) from Arabidopsis thaliana was reverse-transcribed, amplified and expressed under the control of CaMV 35S promoter in transgenic tobacco plants. Several lines were established and tested for the expression of P5CS. Drought and salinity were applied as osmotic stresses and proline content of the transformed plants was compared with that of non-transformed controls. Results indicate that transgenic lines express higher levels of proline and show enhanced resistance to the applied osmotic stress as compared to the non-transgenic plants.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Small Changes in Gene Expression of Targeted Osmoregulatory Genes When Exposing Marine and Freshwater Threespine Stickleback (Gasterosteus aculeatus) to Abrupt Salinity Transfers

Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na⁺/K⁺-ATPase (ATPA13), cystic fibrosis transmembrane regulator (CFTR) and a voltage gated potassium channel gene (KCNH4) and one stress related heat shock protein gene (HSP70)) in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.     

Effect of NaCl and KCl salts on the growth and solute accumulation of the halophyte Atriplex nummularia

Atriplex nummularia plants are able to grow well in the absence of significant amounts of Na⁺. Medium levels of salinity (100 mM NaCl or KCl) did not cause substantial inhibition of growth but increasing concentrations of salt induced a progressive decline in length and weight of the plants. This inhibition was significantly higher in KCl grown plants than in NaCl grown plants. In addition, although it has been proposed that both K⁺ and Na⁺ are involved in the osmotic adjustment of plants in response to high soil salinity, we show that Na⁺ ions contribute more efficiently than K⁺ ions to perform this function. Our results also indicate that most of the osmotic adjustment of the plant was due to the accumulation of inorganic ions. The strong inhibition of Rb⁺ transport caused by internal sodium suggests that this cation could be efficiently used by the plant and, as a consequence, the transport of other monovalent cations is down-regulated.     

Effects of sublethal exposure to copper on diel activity of sea catfish,Arius felis

The effect of sublethal exposure to copper on the diel activity of sea catfish, Arius felis, was examined after 72 h static exposure to copper (0.0, 0.05, 0.1, or 0.2 mg l^–1 ). The locomotor behavior of 28 individual fish was electronically monitored in a 16-compartment rosette tank. Activity was quantified by the number of entries into compartments per hour. Controls and copper-exposed fish (0.05 mg l^–1) behaved similarly after treatment and displayed no significant effects of handling or copper exposure on diel activity. However, fish exposed to 0.1 and 0.2 mg l^–1 copper were hyperactive immediately following exposure; these fish had significantly greater numbers of entries into compartments per hour for entire 24-h monitoring periods (P < 0.01) and for the 12 h of photophase (P < 0.001) and of scotophase (P < 0.001) than fish in the other two experimental groups. The normal diel activity cycle was abolished. Additionally, exposure of fish to copper at 0.1 and 0.2 mg l^–1 resulted in significantly less variability in activity (P < 0.05) compared to control fish and those exposed to 0.05 mg Cu l^–1.     

A review of some physiological and toxicological responses of freshwater fish to acid stress

Synopsis Data relating to the specific effect of low pH on growth of freshwater fishes are ambiguous. Reproductive failure resulting from acid stress appears to be related to an upset in calcium metabolism and to faulty deposition of protein in developing oocytes. It appears that the ’no effect‘ level of pH depression for successful reproduction is around 6.5. Data on behaviorial responses of freshwater fish to acid stress and CO₂ are described. Most fish appear to be indifferent to pH within the range of approximately 10.5 to 5.5 and between 7.4 and 4.5 CO₂ appears to be the main directive factor. In cases of severe acid stress alteration of gill membranes and/or coagulation of gill mucus occurs and death due to hypoxia may result from a lengthening of the water-blood diffusion distance. Several reports agree that acid stress causes an upset of electrolyte homeostasis in fish but effects of low pH on osmotic permeability are largely lacking. Most hatcheryreared salmonids can tolerate pH 5.0 indefinitely but below this level the homeostatic electrolyte and osmotic regulatory mechanisms become inadequate. When fish are subjected to debilitating acid stress blood pH decreases possibly as the result of flux of H⁺ ions across gill membranes into the blood. This could change transepithelial potential and allow a blood, to-water diffusion of Na⁺ ions down an electrochemical gradient. Lowered ambient pH may interfere with gill calcium levels increasing permeability to both H⁺ and Na⁺ ions or an acidemia may occur as the result of a decrease in the excretion of metabolically produced H⁺ ions and CO₂. When the capacity of the buffer mechanisms is exceeded the blood pH drops and the capacity of hemoglobin to transport oxygen is decreased.     

Differential Responses of Nitrogen-Fixing Cyanobacteria to Salinity and Osmotic Stresses

Two nitrogen-fixing Anabaena strains were found to be differentially tolerant to salinity and osmotic stresses. Anabaena torulosa, a brackish-water, salt-tolerant strain, was relatively osmosensitive. Anabaena sp. strain L-31, a freshwater, salt-sensitive strain, on the other hand, displayed significant osmotolerance. Salinity and osmotic stresses affected nitrogenase activity differently. Nitrogen fixation in both of the strains was severely inhibited by the ionic, but not by the osmotic, component of salinity stress. Such differential sensitivity of diazotrophy to salinity-osmotic stresses was observed irrespective of the inherent tolerance of the two strains to salt-osmotic stress. Exogenously added ammonium conferred significant protection against salinity stress but was ineffective against osmotic stress. Salinity and osmotic stresses also affected stress-induced gene expression differently. Synthesis of several proteins was repressed by salinity stress but not by equivalent or higher osmotic stress. Salinity and osmotic stresses induced many common proteins. In addition, unique salt stress- or osmotic stress-specific proteins were also induced in both strains, indicating differential regulation of protein synthesis by the two stresses. These data show that cyanobacterial sensitivity and responses to salinity and osmotic stresses are distinct, independent phenomena.     

Growth attributes,biochemical modulations,antioxidant enzymatic metabolism and yield in Brassica napus varieties for salinity tolerance

Improvement in salinity tolerance of plants is of immense significance as salt stress particularly threatens the productivity of agricultural crops. This study was designed to assess the tolerance level of six Brassica napus varieties (Super, Sandal, Faisal, CON-111, AC Excel and Punjab) under different levels of salinity (0, 50, 100, 150 & 200 mM) with three replications under CRD. Salt induced osmotic stress curtailed the plant growth attributes, photosynthetic pigments and disturbed ionic homeostasis (K⁺, Na⁺, Ca^2+, Cl^-) but least disturbance as compared to control was found in Super and Sandal cultivars. Punjab canola and AC Excel canola cultivars were least tolerant to salinity because these displayed greater decline in all growth and biochemical attributes. Plants subjected to NaCl induced stress exhibited considerable decline in all attributes under study with proline as exception. Antioxidants (CAT, SOD & POD) showed an obvious change in Canola plants under stress, but greatest decline was displayed at 200 mM NaCl level in all six cultivars. Over all these attributes presented a comparatively stable trend in super and sandal cultivars. This shows presence of physiological resilience and metabolic capacity in these two cultivars to tackle salinity. Similarly, all yield attributes displayed adverse behavior under 150 mM & 200 mM salinity stress. Our results demonstrated that Super and Sandal cultivars of Brassica napus exhibit good performance in salinity tolerance and can be good option for cultivation in salt affected areas.     

Osmotic and ion regulation in different species of acipenserids (acipenseriformes, acipenseridae)

Species specificity of osmotic and ion regulation in five species of acipenserids connected in varying degrees with sea conditions is investigated. The objects of study were freshwater middle-Volga sterlet Acipenser ruthenus; Siberian sturgeon A. baerii, inhabiting the Lena River and migrating to the sea near the river mouth; and the following migratory acipenserids of the Volga-Caspian Basin (regular diadromous migrations): the Russian sturgeon A. gueldenstaedtii, the great sturgeon Huso huso, and the starred sturgeon A. stellatus. The study was carried out on immature specimens (of age 2+). The level of osmolarity and concentrations of cations (Na⁺, K⁺, Ca²⁺, and Mg²⁺) were determined in blood serum, urine, and liquid from the cavity of spiral valve in fish from the fresh water (control) and after their adaptation (period of seven to ten days) to sea water with salinity 12.5–12.7 and 18‰. The increase was found in the functional activity of the mechanism of osmotic and ion homeostasis in sturgeons according to the increase of the environmental salinity of a particular species. The freshwater sterlet adapted to the sea environment as osmoconformer. The other studied species were capable of maintaining a relative stability of osmolarity and ion concentration in blood serum at different levels while adapting to the sea environment; they changed from hyperosmotic (in fresh water) to hypoosmotic type of regulation. The kidney plays a more significant role in the removal of excessive Na⁺ in sturgeons than in bony fish. An increase in the sodium-reabsorption and magnesium-secretion functions of the kidney as well as the sodium-absorption function of the intestines was observed in sturgeons, relative to the increase of environmental salinity.     

Salinity tolerance in diapausing embryos of the annual killifish <Emphasis Type="Italic">Austrofundulus limnaeus</Emphasis> is supported by exceptionally low water and ion permeability

The annual killifish Austrofundulus limnaeus inhabits rainwater pools in the Maracaibo basin of Venezuela. This species persists in ephemeral habitats by producing diapausing embryos that are resistant to the stresses imposed by the drying of their aquatic habitat. Embryos of A. limnaeus are likely exposed to a highly variable osmotic environment during development, but their tolerance of osmotic stress has not been characterized. We investigated the capacity of these embryos to survive in hypersaline environments and evaluated the possible mechanisms used to support osmoregulation. Diapausing embryos of A. limnaeus defend their internal osmolality of around 290 mOsmol kg⁻¹ H₂O⁻¹ against salt stress as high as 50 ppt salinity. We find that diapausing embryos of A. limnaeus have a permeability to water that is orders of magnitude lower than other teleost fish embryos. The activity of ion motive ATPases that may be important in the extrusion of ions via mitochondrial rich cells do not appear to be playing a large role in osmoregulation of A. limnaeus embryos. We conclude that for the duration of embryonic development the unique properties of the enveloping cell layer of A. limnaeus embryos acts as a permeability barrier to water and ions and supports osmoregulation in this species in response to a broad range of osmotic environments. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reaction of osmoregulatory system in juveniles of Russian sturgeon <Emphasis Type="Italic">Acipenser gueldenstaedtii</Emphasis> (Acipenseridae) on influence of comatotropin of carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

For the first time by the example of juveniles of Russian sturgeon Acipenser gueldenstaedtii Brandt, the role of growth hormone of hypophysis in the osmotic regulation of acipenserids was studied, also the process of transformation of reserve chloride was described. In Russian sturgeon juveniles, administration of growth hormone of carp Cyprinus carpio at a dose of 1 mg per 1 g body weight causes changes in the fine structure of numerous reserve (undifferentiated) gill epithelial cells and thus stimulates their transformation into mature chloride cells involved in transport of monovalent ions. Increase in the number of chloride cells, on account of reserve cells, was accompanied by higher enzymatic activity of Na⁺/K⁺-ATPase in gill homogenates of the injected fish adapting to a hyperosmotic environment of salinity 12.5‰ (403 mosm/l) than in the intact fish under the same conditions but without hormonal treatment. Morphophysiological changes observed in the injected juveniles provided the more efficient dynamics of blood serum osmolarity under saline exposure than that in the intact fish. Their serum osmolality during the maximum rise (24 hours after the transfer of fish into the water of 12.5‰ salinity) was lower (p <0.01), than that of the intact fish. The inclusion of growth hormone in the osmoregulatory process was established.     

Contributions of inorganic ions,soluble carbohydrates,and multiatomic alcohols to water homeostasis in <Emphasis Type="Italic">Artemisia lerchiana</Emphasis> and <Emphasis Type="Italic">A. pauciflora</Emphasis>

Two morphological forms of wormwood Artemisia lerchiana (f. erecta and f. nutans) and A. pauciflora Web. (morphological form erecta) were grown on sand culture at a range of NaCl concentrations in the nutrient medium and then assayed for Na⁺, K⁺, and Cl^? content in various organs. In addition, the content of mono-, di-, and trisaccharides and multiatomic alcohols (mannitol and glycerol); water content; and organ biomass were determined. All plants examined showed high NaCl tolerance, comparable to that of halophytes. They were able to maintain high tissue hydration under conditions of salinity-induced growth suppression. The intracellular osmotic pressure in wormwood organs was mainly determined by the presence of Na⁺, K⁺, and Cl^?, as well as by mono-, di-, and trisaccharides, mannitol, and glycerol. The high content of Na⁺ and Cl^? in wormwood organs was also observed in the absence of salinity, which implies the ability of these organs to absorb ions from diluted NaCl solutions and accumulate ions in cells of their tissues. With the increase in salinity, the content of Na⁺ and Cl^? in roots and leaves increased to even higher levels. It is concluded that the ability of wormwood plants to absorb and accumulate inorganic ions provides for sustainable high intracellular osmotic pressure and, accordingly, low water potential under drought and salinity conditions. Growing plants under high salinity lowered the content of monosaccharides in parallel with accumulation of the trisaccharide raffinose. It is supposed that soluble carbohydrates and multiatomic alcohols are not only significant for osmoregulation but also perform a protective function in wormwood plants. The lower osmotic pressure in root cells compared to that in leaf cells of all plants examined was mainly due to the gradient distribution of K⁺ and Cl^? between roots and leaves. The two Artemisia species and two morphological forms of A. lerchiana did not differ appreciably in the ways of water balance regulation. It is found that different morphologies of two A. lerchiana forms are unrelated to variations in intracellular osmotic and turgor pressures.     

盐度渐变与骤变对脊尾白虾渗透、代谢及免疫相关酶活力的影响

为探讨盐度变化对脊尾白虾(Exopalaemon carinicauda)渗透、代谢及免疫相关酶活力的影响,实验设置了盐度渐变和骤变两个实验。渐变实验,设置5、10、15、20、25、30、33(CK)、40和45共9个盐度梯度;骤变实验,盐度从33突变至0、5、15、25和45,检测血清ATP酶(包括Na+/K+-ATP酶和总ATP酶)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)及超氧化物歧化酶(SOD)活力。结果表明,渐变情况下,盐度为5时,ATP酶活力出现最高值,然后随着盐度的升高表现出先降低后升高的趋势。总ATP酶活力在盐度为15—30之间较稳定,并在此范围内达到最低值。AKP和ACP活力几乎不受盐度渐变的影响。SOD活力随盐度的升高,先上升后下降,并在盐度为33时达到最大值。骤变情况下,ATP酶活力随时间波动较大,AKP和SOD随时间波动较小,而ACP几乎不受影响。结果说明,盐度骤变对脊尾白虾酶活力的影响较盐度渐变明显,ATPase和SOD活力更易随盐度而变化,代谢酶(AKP、ACP)受盐度变化的影响较小,说明渗透调节和免疫相关酶活力对盐度变化反应敏感,养殖过程中应尽量保持盐度稳定。     

Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent) : transition metal ion requirement for activity and sulfhydryl group reactivity

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phosphoenolpyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg²⁺ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co²⁺ > Mn²⁺ > Cd^u2+ > Ni²⁺ ⪢ Fe²⁺ > VO²⁺, while Zn²⁺ and Ca²⁺ had no activating effects. When we investigated the effect of varying the type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO₂ substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn²⁺ activated enzyme the inhibition was purely non-competitive (K_ii = K_is) towards all substrates, while for the Co²⁺-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5′-dithiobis(2-nitro-benzoate) (DTNB) lead to an almost complete inhibition of Mn²⁺-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO₃⁻ had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co²⁺-activated enzyme than for its Mn²⁺-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn²⁺ and ADP afforded almost complete protection from inactivation; in the presence of Co²⁺ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.