Rafael Torronteras

Acute toxicity of manganese in goldfish Carassius auratus is associated with oxidative stress and organ specific antioxidant responses.

Manganese is a relatively common, yet poorly studied element in freshwater ecosystems, where it can be significantly bioconcentrated. The knowledge about the mechanisms of Mn toxicity on fish health is still limited. The aim of the present study was to assess the potential induction of oxidative stress and the antioxidant response after a 96 h waterborne Mn-exposure (at 0.1 and 1mM) in gill, kidney, liver and brain of goldfish (Carassius auratus). Mn 1mM induced an increase of lipid hydroperoxides, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in all tissues with the exception of SOD inhibition in the brain. Particular response of catalase (CAT) was indicated-its inhibition in the liver and kidney, but activation in the gill. Exposure to Mn 0.1mM provoked most prominent changes in the liver and did not change the indexes in brain. These results strongly suggest that Mn exposure caused a generalized oxidative stress in the fish and revealed an organ specific antioxidant response involving a differential modulation of the SOD, CAT and GPx activities.

IDENTIFICATION AND PHYSIOLOGICAL ASPECTS OF A NOVEL CAROTENOID-ENRICHED, METAL-RESISTANT MICROALGA ISOLATED FROM AN ACIDIC RIVER IN HUELVA (SPAIN)1

A heavy‐metal‐resistant, carotenoid‐enriched novel unicellular microalga was isolated from an acidic river in Huelva, Spain. The isolated ribosomal 18S subunit rDNA sequence showed homology with known sequences from green microalgae, the closest sequence (98% homology) belonging to the genus Coccomyxa. The isolated microalga therefore was an up to now uncultured microalga. The microalga was isolated from Tinto River area (Huelva, Spain), an acidic river that exhibits very low pH (1.7–3.1) with high concentrations of sulfuric acid and heavy metals, including Fe, Cu, Mn, Ni, and Al. Electron micrographs show that the microalga contains a large chloroplast with a presence of lipid droplets, an increased number of starch bodies as well as electron‐dense deposits and plastoglobules, the last observed only in iron‐exposed cells. Unlike other acidophile microalgae, the isolated microalga showed high growth rates when cultivated photoautotrophycally (up to 0.6 d−1) in a suitable culture medium prepared at our laboratory. The growth was shown to be iron dependent. When the microalga is grown in fluidized bed reactors, the high growth rates resulted in unexpectedly high productivities for being a microalga that naturally grows in acidic environments (0.32 g·L−1·d−1). The microalga also grows optimally on reduced carbon sources, including glucose and urea, and at an optimal temperature of 35°C. The alga pigment profile is particularly rich in carotenoids, especially lutein, suggesting that the microalga might have potential for antioxidant production, namely, xanthophylls.

Different effects of somatostatin on in vitro growth hormone release in two porcine breeds with different growth rates.

A perifusion system of anterior pituitary (AP) tissue was used to investigate the temporal interaction of growth hormone‐releasing factor (GRF) and somatostatin (SRIF) in the control of GH secretion in two pig breeds, Göttingen Miniature Pig (GMP), a small obese breed, and German Landrace (GLR), a conventional lean breed. AP tissue pieces derived from sexually mature ovariectomized animals were perifused (6 replicates per treatment) and fractions were collected at 10 min intervals. Basal GH release (ng‐mP‐1 mg‐1 AP) in GLR was twice that of GMP (P < 0.001). Exposure to 10 min pulses of 1 nM GRF repeated 3 times at 2 h intervals resulted in rapid stimulatory GH responses (area under the curve) which became attenuated (P < 0.05) over time in GMP but not in GLR. Surprisingly, during and following the exposure of AP tissue from GMP to 10‐, 20‐, or 40‐min pulses of 10 nM SRIF alone, GH release was markedly stimulated (P < 0.05), while AP tissue from GLR only showed a weak rebound GH release after SRIF pulses. With AP tissue from GLR low concentrations (0.1 nM SRIF) amplified GRF‐induced GH release, whereas 1 nM or 10 nM SRIF inhibited GRF‐induced GH release. However, concomitant exposure of AP tissue from GMP to 0.1, 1 or 10 nM SRIF during a GRF pulse markedly enhanced the GH response (P < 0.05), compared to 1 nM GRF alone, except for 1 nM SRIF which inhibited the GH response to the first GRF pulse. Thus the presence of SRIF, and not only its withdrawal, is an important factor in setting the timing and duration of GH pulses in both breeds. In GLR the concentration of SRIF is more important than the duration and/or type of SRIF pulse. In contrast, in GMP type and/or duration of SRIF pulses seem to be crucial to optimize pulsatile GH release and even determine peak height of GH pulses caused by GRF. These findings indicate clear breed differences in the role of SRIF and in the control of GH release by the interplay of GRF and SRIF. The ‘paradoxical’ effect of SRIF suggests that the role of SRIF is much more complex than that of a mere inhibitor and whose real role could be a modulator either of GH pulse and/or GRF action on GH release.

Effect of Selenate on Viability and Selenomethionine Accumulation of Chlorella sorokiniana Grown in Batch Culture

The aim of this work was to study the effect of Se(+VI) on viability, cell morphology, and selenomethionine accumulation of the green alga Chlorella sorokiniana grown in batch cultures. Culture exposed to sublethal Se concentrations of 40 mg·L−1 (212 μM) decreased growth rates for about 25% compared to control. A selenate EC50 value of 45 mg·L−1 (238.2 μM) was determined. Results showed that chlorophyll and carotenoids contents were not affected by Se exposure, while oxygen evolution decreased by half. Ultrastructural studies revealed granular stroma, fingerprint-like appearance of thylakoids which did not compromise cell activity. Unlike control cultures, SDS PAGE electrophoresis of crude extracts from selenate-exposed cell cultures revealed appearance of a protein band identified as 53 kDa Rubisco large subunit of Chlorella sorokiniana, suggesting that selenate affects expression of the corresponding chloroplast gene as this subunit is encoded in the chloroplast DNA. Results revealed that the microalga was able to accumulate up to 140 mg·kg−1 of SeMet in 120 h of cultivation. This paper shows that Chlorella sorokiniana biomass can be enriched in the high value aminoacid SeMet in batch cultures, while keeping photochemical viability and carbon dioxide fixation activity intact, if exposed to suitable sublethal concentrations of Se.

Different exocytotic morphology in amphibian prolactin and growth hormone cells stimulated in vitro with TRH

Exocytotic process in growth hormone (GH) and prolactin cells (PRL) of the frog anterior pituitary have been examined using an experimental design that has been previously demonstrated to increase the release of hormone from both cell types. Hemipituitaries of the same animals were superfused either with medium alone or containing 100 ng/ml of thyrotropin-releasing hormone (TRH) for 24 hr. PRL and GH cells were identified by the colloidal gold method using anti-human prolactin and anti-ovine growth hormone as primary antisera. In hemipituitaries cultured with medium alone, PRL and GH cells showed few exocytotic figures with different morphology in both cells types. In TRH treated hemipituitaries, PRL cells showed numerous exocytotic vacuoles containing immunoreactive granulated material that was preferentially located near basal lamina. On the other hand, GH cells showed higher amount of exocytotic vacuoles containing heterogeneous immunoreactive material, located along the cell membrane. In PRL cells single secretory granules are secreted, whereas GH cells showed multigranular exocytosis. These results indicate that in PRL and GH amphibian cells exocytotic process has a different polarity and morphology and that this process increases with TRH stimulation.

Erratum to: Phylogenetic characterization and morphological and physiological aspects of a novel acidotolerant and halotolerant microalga Coccomyxa onubensis sp. nov. (Chlorophyta, Trebouxiophyceae)

The genus Coccomyxa comprises green microalgae, which can be found worldwide in remarkably versatile aquatic and terrestrial ecosystems including symbiotic associations with a number of different hosts. In this study, we describe a new species, Coccomyxa onubensis, based on 18S and ITS ribosomal DNA (rDNA) sequence data. Coccomyxa onubensis was isolated from acidic water, and its ability to adapt to a wide range of acidic and alkaline pH values and to high salinity was analyzed. The long-term adaptation capacity of the microalga to such extreme conditions was evaluated by performing continuous repeated batches at selected salt concentrations and pH values. Adapted cultures of C. onubensis were found to yield high biomass productivities from pH 2.5 to 9, with maximum yields at acidic pH between 2.5 and 4.5. Moreover, C. onubensis was also found to adapt to salinities as high as 0.5 M NaCl, reaching biomass productivities that were similar to those of control cultures. Ultrastructural analysis by transmission electron microscopy of C. onubensis cells adapted to high salinity showed a robust response to hyperosmotic shock. Thus, C. onubensis was found to be acidotolerant and halotolerant. High biomass productivity over a wide range of pH and salinities denotes C. onubensis as an interesting candidate for various biotechnological applications including outdoor biomass production.

Differential Ontogenetic Patterns of in vitro Desensitization to GHRH in Fetal and Neonatal Anterior Pituitary

The aim of this study was to assess the ontogenetic changes in vitro in both the responsiveness of anterior pituitary tissue to growth hormone-releasing hormone (GHRH) and the critical role of GHRH in the long-term regulation of pulsatile GH secretion during perinatal porcine life. A superfusion system was used to apply three consecutive 10-min pulses of GHRH (the first of 1 nM and the other two of 10 nM) for 3 consecutive days in pituitary glands isolated from fetal (95- and 110-day) and neonatal (12-day) male pigs. In fetuses, total GHRH-induced GH release decreased progressively over the 3 days. However, in neonates, GH did not decrease until day 3, but remained higher than in fetuses. When each GH pulse was assessed individually, fetuses showed a similar pattern. GH secretion induced by the first GHRH pulse on days 1 and 2 was lower than that induced by the second and third pulses. By day 3, GH release lowered dramatically after all pulses. In contrast, in neonates no differences were observed among the GH levels induced by the three GHRH pulses at any day, although day 3 showed lower GH rates. In conclusion, during perinatal development, a desensitizing effect to long-term repetitive GHRH pulses was observed in both fetuses and neonates, but this effect was delayed in neonates. Thus, the capacity of somatotrope cells to maintain GH response to GHRH seems to be developmentally regulated during perinatal stages. Furthermore, the frequency of GHRH pulses, rather than the concentrations, might be a key factor to elicit desensitization.