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.

     

Decrease in occurrence of fast startle responses after selective Mauthner cell ablation in goldfish (Carassius auratus )

A single action potential in one of a pair of reticulospinal neurons, the Mauthner cells, precedes a short-latency electromyographic response of the trunk and tail musculature on the opposite side of the body and a fast startle response in goldfish. It has been postulated that not only the Mauthner cell, but also an array of neurons can trigger or participate in fast startle responses (Eaton et al. 1991). We have selectively ablated the Mauthner cells in goldfish to study how neurons of the brainstem fast startle response network interact. The probability of eliciting a fast startle response was significantly less in fish with double Mauthner cell ablations, as compared to the responsiveness of control fish. The finding that there is a significant decrease in the occurrence of fast startle responses in animals with no Mauthner cells, implies that the Mauthner cell may play a role in triggering the involvement of the other network elements in fast startle responses. We hypothesize that Mauthner cell activation may be important in bringing those reticulospinal neurons that are “primed” by the behavioral context to threshold and provides the basis for studies focused on the interactive nature of the brainstem startle response network. Accepted: 4 November 1998     

Effect of cysteine modifications on the activity of the ‘small’ Clostridium perfringens sialidase

The small (43 kDa) sialidase of Clostridium perfringens is inhibited by low concentrations of mercury ions. For the investigation of possible functional roles of the enzyme's four cysteine residues at the amino acid positions 2, 282, 333 and 349, they were separately altered to serine by site-directed mutagenesis. The four mutant sialidases expressed in E. coli and purified by metal chelate chromatography were markedly reduced in specific activity when compared to the wild-type enzyme but with the exception of C282S exhibited similar KM-values indicating an unchanged mode of substrate binding. The substrate specificity was also conserved for C2S, C282S, and C333S. Only the C349S sialidase exhibited a higher relative activity with colominic acid and the 2,6-linked sialic acid of sialyllactose compared to the 2,3-linked isomer than the other mutants. Chemical modifications with the thiol-blocking reagents N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB) and HgCl2 had little effect on the C282S sialidase, e.g., 6% inhibition by 5 m M NEM compared to reductions in activity between 65 and 90% for the wild-type and other mutant enzymes, supporting the idea that among the enzyme's cysteines, Cys-282 has the highest structural or functional significance. The results also explain the higher mercury tolerance of Salmonella typhimurium and Clostridium tertium sialidases, which have the positions equivalent to Cys-282 altered to Val and Thr, respectively, indicating that the thiol group of Cys-282, despite being situated near the active site, is not involved in catalysis.     

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.     

Distribution of fibres reacting with an α-endorphin antiserum in the neurohypophysis of Carassius auratus and Cyprinus carpio

Summary A strong positive immunoreaction with an -endorphin antiserum occurs in two distinct sites of the goldfish and carp neurohypophysis. Fluorescent nerve terminals are found in the laminar nerve processes located in the rostral pars distalis, but the immunocytological reaction is mainly localised on the nerve processes of the posterior neurohypophysis lying between the intermediate lobe cells. Almost all the digitations of the neurohypophysis are strongly fluorescent. The immunoreactive fibres probably originate from the hypothalamus, where perikarya displaying the same immunoreaction have been found in the pars lateralis of the nucleus lateralis tuberis and in some minor centres. The possibility that the immunoreactive substances revealed on the neurohypophyseal processes may originate in the intermediate lobe cells is also discussed. It has now to be established if this hypothalamo-hypophyseal system contains a substance with endorphic properties or only some immunologically related substance devoid of the corresponding physiological activities.     

Modelling the Dynamics of Crystal Meth (‘Tik’) Abuse in the Presence of Drug-Supply Chains in South Africa

Substance abuse remains a global problem, with immense health and social consequences. Crystal meth, known as ‘tik’ in South Africa, is a growing problem, and its supply chains have equally grown due to increased numbers of ‘tik’ users, especially in the Western Cape province of South Africa. We consider a model for ‘tik’ use that tracks drug-supply chains, and accounts for rehabilitation and amelioration for the addicted. We analyse the model and show that it has a unique drug-free equilibrium. We prove that the drug-free equilibrium is globally stable when the reproduction number is less than one. We also consider both slow and fast dynamics, and show that there is a unique drug-persistent equilibrium when the reproduction number exceeds one. The model is fitted to data on ‘tik’ users in rehabilitation in the Western Cape province. A sensitivity analysis reveals that the parameters with the most control over the epidemic are the quitting rate of light-drug users and the person-to-person contact rate between susceptible individuals and ‘tik’ users. This suggests that programs aimed at light-drug users that encourage them to quit will be significantly more effective than targeting hard-drug users, either in quitting or in rehabilitation. Similarly, the person-to-person contact rate may be reduced by social programs that raise awareness of the dangers of ‘tik’ use and discourage light users from recruiting others.     

Detection of ‘Candidatus Cardinium’ Bacteria from the Haploid Host Brevipalpus Californicus (Acari: Tenuipalpidae) and Effect on the Host

Brevipalpus californicus (Banks) was infected with ‘Candidatus Cardinium’ bacteria (Cardinium). Tetracycline-treated females produced many male progeny even though untreated females produced only female progeny. B. californicus appears to be feminized by Cardinium. The values for net reproduction rate (R₀), generation time (T) and intrinsic rate of natural increase (r_m) calculated for B. californicus were 7.48/day, 31.45 days and 0.064/day, respectively. The comparison of infected females with uninfected males and other closely related species, indicated that Cardinium does not have a negative effect on the fitness of B. californicus.     

Effect of busulphan treatment and elevated temperature on the expression of the β-pol gene in rat testis

Changes in the expression pattern of the DNA polymerase gene during inhibition of spermatogenesis by busulphan and by temperature (artificial cryptorchidism) have been studied. Transient arrest of spermatogenesis in two-month-old rats after injection of a single dose of busulphan (10 mg/kg) resulted in parallel but transient decrease in the 1.4 kb of -pol mRNA level to an undetectable value, followed by its reappearance after resumption of spermatogenesis. An artificial cryptorchidism also caused a drastic decrease of -pol mRNA level. Both results as well as morphological examination of testis after busulphan injection and artificial cryptochidism revealed that spermatocytes and spermatids represent the testicular cell fraction containing the elevated amount of -pol mRNA. Involvement of DNA polymerase in meiotic recombination is discussed.     

Effect of liquid culture on the growth and development of miniature rose (Rosa chinensis Jacq. ‘Minima’)

The growth of miniature rose (Rosa chinensis Jacq. Minima) shoots cultured on liquid medium was greater relative to those cultured on two-phase (solid + liquid) medium or solid medium alone. Shoot multiplication ratio (number of multiple shoots per explant per subculture) on liquid medium was higher with 17.8–26.6 M 6-benzyladenine at compared to that at 0–8.9 M. Shoots grown on 30 ml or more of liquid medium had a higher multiplication ratio than those grown on 10 or 20 ml. The growth and multiplication ratio increased when the culture period was extended from 3 to 6 weeks, although plantlets began to exhibit some chlorosis by the 6^th week. These conditions were maintained over four subcultures for cultivars Baby Katie, Lavender Jewel, Red Sunblaze and Royal Sunblaze, with no significant change in multiplication ratio over time.Abbreviations BA 6-benzyladenine - NAA 1-naphthaleneacetic acid     

Observations on the biology of Leuciscus ‘svallize’ in the Kremasta reservoir (Greece)

The age, growth, feeding habits and reproductive biology of Leuciscus svallize in artificial lake Kremasta (Greece), are described. There is a considerable similarity in the feeding and reproductive characteristics between L. svallize and L. cephalus and also a similarity in the life span, growth patterns and age of maturation between L. svallize and some southern L. cephalus populations, which may suggest similar responses of closely related taxa to similar conditions.     

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.     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

Immunolocalization of exoglycoproteins (“ependymins”) in the goldfish brain

Exoglycoproteins (X-GPs) are a group of very abundant soluble glycoproteins in the goldfish, brain. Immunostaining with polyclonal antisera to X-GPs revealed consistent perinuclear staining in the cells of the inner and intermediate layers of the leptomeninx, which is homologous to the piaarachnoid. Immunolabelling was also prominent in the outer wall of capillaries, and in a variable population of 10–12 m granular cells that appeared mainly near the ventricles and occasionally within the ventricles or under the meninges. In some cases, small and medium-sized lymphocytes were immunostained. Lymphocytes were sometimes associated with the granular cells, which may be hematogenous cells in transit toward the ventricles. the choroid plexus, saccus dorsalis, the roof of the third ventricle and Reissner's fiber showed strong immunostaining. The localization of the X-GPs suggests that they may contribute to maintenance of the blood-brain barrier or to regulation of immune function within the brain.Special issue dedicated to Dr. Sidney Ochs.     

‘Good-genes’ and ‘compatible-genes’ effects in an Alpine whitefish and the information content of breeding tubercles over the course of the spawning season

Abies ziyuanensis is a highly endangered fir species endemic to South China. Unlike other Abies species that are distributed in areas with cold climates, A. ziyuanensis is restricted to several isolated island-like localities at subtropical mountains. In this study, we used dominant amplified fragment length polymorphism (AFLP) and co-dominant simple sequence repeats (SSR) markers to infer the genetic structure of A. ziyuanensis. Seven populations consisting of 139 individuals were sampled across their whole distribution. A. ziyuanenesis has a relatively low level of genetic variation, with a mean genetic diversity per population (He) of 0.136 (AFLP) and 0.337 (SSR), which is lower than that of other reported endemic species based on the same kind of marker. We observed high population differentiation, with Gst = 0.482 (AFLP) and Fst = 0.250 (SSR), among the seven populations. AMOVA also detected significant differentiation among populations (Φst (AFLP) = 0.550 and Φst (SSR) = 0.289) and among regions (Φct (AFLP) = 0.139 and Φct (SSR) = 0.135) in both marker types. Both ongoing evolutionary forces (e.g., genetic drift resulting from small population size) and historical events (e.g., population contraction and fragmentation during and after the Quaternary glacial cycles) may have contributed to the genetic structure in A. ziyuanensis.