Acid–base regulation in isolated gill cells of the goldfish (Carassius auratus)

Mechanisms of acid release and intracellular pH (pH_i) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pH_i was determined using the fluorescent probe 2,7-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na⁺ channel and Na⁺/H⁺ exchanger (NHE) inhibitor, had no effect on pH_i, but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH₄Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na⁺. Under steady-state conditions, 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid (DIDS), a HCO₃^–-transport inhibitor, caused a slow acidification of pH_i, and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A₁, an inhibitor of V-ATPase, had no effect on steady-state pH_i and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na⁺-dependent HCO₃^– transport is the dominant mechanism besides an NHE and V-ATPase to control pH_i in goldfish gill cells.Communicated by G. Heldmaier     

Effects of photoperiod on plasma corticoid levels in the goldfish,Carassius auratus—role of the pineal

• 1.1. The effects of photoperiod and pinealectomy on plasma corticoid levels in the goldfish (Carassius auratus) were examined.
• 2.2. Plasma corticoid levels differed in goldfish maintained under different photoperiod regimes, but this response varied seasonally.
• 3.3. Pinealectomy altered the effects of photoperiod on plasma corticoid levels but this effect varied with season.
• 4.4. Plasma corticoid levels were correlated with ovarian activity. The effects of photoperiod on plasma corticoid levels appear to be related to the influence of light on reproduction.
• 5.5. The alteration of plasma corticoid levels in pinealectomized fish may be due to the role this organ plays in mediating photoperiod effects on gonadal activity.

     

Molecular cloning and differential expression patterns of the regulatory subunit B′ gene of PP2A in goldfish, Carassius auratus

It is well established that the protein serine/threonine phosphatase 2A (PP2A) plays very important roles in many different cellular processes, including cell proliferation and differentiation, gene expres-sion, neurotransmission, apoptosis, and aging. PP2A consists of three heterogenic subunits: the scaffold subunit A, the catalytic subunit C, and the regulatory subunit B. While both the scaffold and the catalytic subunits contain only two forms, at least four families of the regulatory subunits, B, B, B′′, and B′′′ have been identified. These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A. To study the functions of the regulatory subunits of PP2A in lower vertebrates, we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit B′δ of PP2A from gold fish, Carassius auratus using 3′-RACE and 5′-RACE cloning strategies. Our results revealed that the full-length B′δ cDNA contains 2415 bp and encodes a protein of 555 amino acids. The B′δ protein displays a very high level of sequence identity with the B′δ regulatory subunit from other species of vertebrates. Regarding its expression pattern, RT-PCR revealed that the highest level of mRNA was detected in brain, a less level detected in liver, spermary, ovary, kidney and gill, and the lowest level detected in the fin. During different developmental stages of gold fish, the highest level of mRNA expression was detected at the stages of two-cell, mul-tiple-cell, blastula and gastrula, and a decreased level of B′δ ?mRNA was detected in other develop-mental stages. At the protein level, the highest expression level of B′δ protein was found in spermary, ovary, brain and heart, a less amount found in liver and the lowest level detected in kidney, gill and fin. Developmentally, B′δ protein was strongly expressed at the stages of two-cell, multiple-cell, blastula, gastrula, neurula, and optic vesicle, and then decreased at the stages of brain differentiation and eye pigmentation. These results suggest that B′δ appears to play a very important role during gold fish development and also in adult tissue homeostasis.     

Effect of temperature on locomotor activity in the goldfish (Carassius auratus) and the bluegill (Lepomis macrochirus): Presence of an ‘activity well’ in the region of the final preferendum

Goldfish (Carassius auratus) and bluegill sunfish (Lepomis macrochirus) were placed in aquaria where their locomotor activity was monitored by photocells, and tested at various acclimation temperatures over a range encompassing their final thermal preferenda. Activity was pooled over 24-hour periods to eliminate any circadian rhythm effects. Both species exhibited an activity well of reduced locomotor activity in the region of the final preferendum. Goldfish, tested either singly or in groups of 2–5 individuals, exhibited a social-interaction effect which became more pronounced at higher temperatures. These results are discussed in relation to a thermokinetic interpretation of thermo-regulatory behavior in fishes, and to the correspondence between thermal preferenda and thermal optima.     

Anaerobic metabolism of goldfish,Carassius auratus (L.): Ethanol and CO2 excretion rates and anoxia tolerance at 20, 10 and 5°C

• 1.1. Goldfish acclimated to 20, 10 and 5°C were exposed to anoxia. The lethal thresholds expressed as median lethal time (LT₅₀) were found to be 22, 65 and 45 hr respectively.
• 2.2. During anoxia excretion of CO₂ and ethanol was monitored. CO₂/ethanol ratios were initially far over unity, falling to lower values after longer exposures.
• 3.3. The quantitative aspects of ethanol production seem incompatible with the availability of glycogen as a carbon source. Protein is suggested as a possible substrate.

     

Reciprocal inhibiting interactive mechanism between the estrogen receptor and aryl hydrocarbon receptor signaling pathways in goldfish (Carassius auratus) exposed to 17β-estradiol and benzo[a]pyrene

In the aquatic environment, both the estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) responses are established biomarkers for assessing exposure to pollutants. These receptor responses can also be affected by the presence of other classes of pollutants and may result in misinterpretation of existing pollution. In this study, we investigated the interaction between ER-vitellogenin (VTG) and AhR-cytochrome P450 1A (CYP1A) signaling pathways in goldfish (Carassius auratus) after 10 days exposure to pollutants. 17β-Estradiol (E(2)) and benzo[a]pyrene (BaP) were selected as the ER and AhR agonists, respectively. The messenger RNA (mRNA) expression of ER-VTG and AhR-CYP1A in liver was determined using quantitative real-time polymerase chain reaction (QRT-PCR). VTG, endogenous E(2) and 7-ethoxyresorufin-O-deethylase (EROD) were also studied. Exposure to E(2) and BaP alone significantly induced the gene expression of ERα-VTG and AhR2-CYP1A, respectively. Moreover, the obvious expression of related proteins was also observed. However, these inductions were significantly reduced after combined exposure to E(2) and lower concentrations of BaP (20 and 50 μg/L), indicative of a reciprocal inhibiting ER-AhR interaction. However, high concentrations (100 μg/L) of BaP did not affect the E(2)-induced gene expression. Changes in VTG protein were in accordance with the expression of VTG mRNA, and more VTG protein was observed in liver than in serum. The induced endogenous E(2) levels were suppressed by the presence of BaP. While the gene expression of CYP1A showed a concentration-dependent increase, EROD induction exhibited a bell-shaped concentration-response curve. Taken together, these results demonstrate a reciprocal inhibiting mode of ER-AhR interactions and may lead to a possible underestimation of actual exposure.     

Physiological responses to cold (10° C) in men after six months' practice of yoga exercises

A study was conducted on 30 healthy soldiers (age: 40–46 years) to assess the effect of selected yogic exercises (asanas) on some physiological responses to cold exposure. They were randomly divided into two groups of 15 each. One group performed regular physical exercises of physical training (PT), while the other group practised yogic exercises. At the end of 6 months of training, both the groups were exposed together to cold stress at 10°C for 2 h, and the following parameters were periodically monitored during cold exposure: heart rate (fH), blood pressure (BP), cardiac output , oral temperature (T_or), skin temperature (T _sk), respiratory rate (fR), minute ventilation , oxygen consumption , and shivering response by integrated electromyogram (EMG). There were progressive increases inBP, fR, , , and and decreases infH,T _or andT _sk during cold exposure in both the groups. However, the decrease inT _or and the increases in and were relatively lower (P<0.01) in the yoga group as compared to the PT group. The shivering response appeared much earlier and was more intense in the PT group. These findings suggest that practice of yoga exercises may improve cold tolerance.     

Assessment of Possible Causes of Changes in Аbundance and Sexual Structure in Populations of Prussian Carp (<Emphasis Type=Italic>Carassius auratus gibelio</Emphasis> Bloch., 1783)

The prevalence of processes of the increase in abundance and changes in the sexual structure of Prussian carp populations in waterbodies differing dramatically in ecological properties and distant from each other (Lake Sevan and Rybinsk Reservoir) indicates the presence of global factors affecting climatic zones. The increase in environmental temperature due to global warming may be such a factor. Experimentally proven high thermophilicity and the thermal stability of Prussian carp, as well as its low sensitivity to hypoxia, provide some advantages for this species over many other fish inhabiting the same waterbodies.     

Monocrotophos Pesticide Decreases the Plasma Levels of Total 3,3′,5-Triiodo-l-Thyronine and Alters the Expression of Genes Associated with the Thyroidal Axis in Female Goldfish (Carassius auratus)

Our recent study showed that monocrotophos (MCP) pesticide disrupted the hypothalamic-pituitary-thyroid (HPT) axis in male goldfish (Carassius auratus); however, the effects of MCP on the thyroid system in female goldfish are remain unclear. In the present study, plasma thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels were evaluated in female goldfish exposed to 0.01, 0.10, and 1.00 mg/L of 40% MCP-based pesticide for 21 days in a semi-static exposure system. Expression profiles of HPT axis-responsive genes, including transthyretin (ttr), deiodinases (d1, d2, and d3), tshβ, thyrotropin-releasing hormone (trh), and corticotrophin-releasing hormone (crh), were determined. The results indicated that MCP decreased the plasma levels of total 3,3′,5-triiodo-l-thyronine (TT₃) and the ratio of TT₃ to total 3,3′,5,5′-l-thyroxine (TT₄), and induced alternative expression of TH-related genes. Exposure to 0.01 and 0.10 mg/L MCP pesticide resulted in the up-regulation of ttr mRNA. The reduction of plasma TT₃ levels was partly attributed to an increase in the metabolism of T₃ in the liver, as revealed by the highly elevated hepatic d1 and d3 mRNA levels in the MCP treatment groups, and the expression of hepatic d3 showed a negative correlation with the plasma TT₃/TT₄ levels in females. Moreover, the plasma TSH levels were lower in females exposed to 0.01 and 0.10 mg/L MCP pesticide, whereas the up-regulation of tshβ mRNA levels was compensated by the decreased plasma TT₃ levels. These results indicated that MCP had the potential to influence several pathways of HPT axis homeostasis in female goldfish.     

Genetic polymorphisms of Fas (CD95) and Fas ligand (CD178) influence the rise in CD4+ T cell count after antiretroviral therapy in drug-naïve HIV-positive patients

Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus. To investigate whether the genetic background of the host plays a role in the immunoreconstitution, we studied the influence of different Fas and FasL polymorphisms on CD4+ T lymphocyte count and plasma viral load following initiation of highly active antiretroviral therapy (HAART) in drug-naïve HIV+ patients. We studied 131 individuals, who were compared to 136 healthy donors. Statistical analysis was performed by using X ² test, Fischer's Exact Test, and analysis for repeated measurements. The group of HIV+ patients had an unexpected lower frequency of FasLnt169 polymorphism (delT allele) than healthy controls (p=0.039). We then observed no significant differences in the immune reconstitution, in terms of CD4+ T cell increase, when the influence of single alleles of the gene Fas or FasL was considered. However, the combination of some polymorphisms of Fas or FasL significantly influenced CD4+ T cell production and viral load decrease, showing that these genes can play a role in the immunoreconstitution triggered by antiretroviral therapy.