Two cholecystokinin receptor subtypes are identified in goldfish, being the CCKAR involved in the regulation of intestinal motility.

Cholecystokinin (CCK) plays a key role in the digestive physiology of vertebrates. However, very little is known about the role of CCK on intestinal functions in fish. The present study identifies two CCK receptor subtypes in a stomachless teleost, the goldfish (Carassius auratus), and investigates by using an in vitro system their involvement mediating the effects of the sulfated octapeptide of CCK (CCK-8S) on the motility of isolated proximal intestine. Partial-length mRNAs encoding two CCK receptor isoforms (CCKAR and CCKBR.I) were sequenced and the structural analysis showed that both receptors belong to the G-protein coupled receptor superfamily. Both goldfish CCK receptor sequences were more closely related to zebrafish sequences, sharing the lowest similarities with cavefish and tilapia. The highest expression of goldfish CCKAR was observed along the whole intestine whereas the CCKBR gen was predominantly expressed in the hypothalamus, vagal lobe and posterior intestine. Application of CCK-8S to the organ bath evoked a concentration-dependent contractile response in intestine strips. The contractions were not blocked by either tetrodotoxin or atropine, suggesting that CCK-8S acts on the gut smooth muscle directly. Preincubations of intestine strips with devazepide and L365,260 (CCKAR and CCKBR receptor selective antagonists) showed that the CCK-8S-induced contraction could be partially mediated by the CCKAR receptor subtype, which is also the most abundant CCK receptor found in gastrointestinal tissues. In conclusion, two CCK receptors with a differential distribution pattern has been identified in goldfish, and the CCKAR subtype is mainly involved in the regulation of intestinal motility by the CCK-8S.

Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free-living micro-oxic and symbiotic conditions.

AIM: In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free-living and symbiotic conditions have been carried out. METHODS AND RESULTS: Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD-dependent oxidase activity when cells were incubated under micro-oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane-bound c-type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb3 oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N-free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation. CONCLUSIONS: The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro-oxic conditions as well as in the expression of the FixO and FixP components of the cbb3 oxidase present in free-living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb3 oxidase operating in free-living micro-oxic cultures and in bacteroids of E. meliloti have been identified.

Enhanced expression of Rhizobium etli cbb3 oxidase improves drought tolerance of common bean symbiotic nitrogen fixation.

To investigate the involvement of Rhizobium etli cbb(3) oxidase in the response of Phaseolus vulgaris to drought, common bean plants were inoculated with the R. etli strain, CFNX713, overexpressing this oxidase in bacteroids (cbb(3)(+)) and subjected to drought conditions. The negative effect of drought on plant and nodule dryweight, nitrogen content, and nodule functionality was more pronounced in plants inoculated with the wild-type (WT) strain than in those inoculated with the cbb(3)(+) strain. Regardless of the plant treatment, bacteroids produced by the cbb(3)(+) strain showed higher respiratory capacity than those produced by the WT strain. Inoculation of plants with the cbb(3)(+) strain alleviated the negative effect of a moderate drought on the respiratory capacity of bacteroids and the energy charge of the nodules. Expression of the FixP and FixO components of the cbb(3) oxidase was higher in bacteroids of the cbb(3)(+) strain than in those of the WT strain under all experimental conditions. The decline in sucrose synthase activity and the decrease in dicarboxylic acids provoked by moderate drought stress were more pronounced in nodules from plants inoculated with the WT strain than in those inoculated with the cbb(3)(+) strain. Taken together, these results suggest that inoculation of plants with a R. etli strain having enhanced expression of cbb(3) oxidase in bacteroids reduces the sensitivity of P. vulgaris-R. etli symbiosis to drought and can modulate carbon metabolism in nodules.

Characterization of two different melatonin binding sites in peripheral tissues of the teleost Tinca tinca.

The aim of the present study was to localize and characterize 2-iodo-melatonin ([(125)I]Mel) binding sites in peripheral tissues of the teleost Tinca tinca. A wide distribution of [(125)I]Mel binding sites in peripheral locations of the tench is found, with highest densities being measured in the heart, gills and kidney, and low density of [(125)I]Mel binding sites in gastrointestinal tract, spleen, liver and gonads. Saturation, kinetics, and pharmacological approaches revealed the presence of, at least, two different [(125)I]Mel binding sites in the tench peripheral tissues. The unique characterized subtype in the heart fulfils all the criteria for a canonical melatonin receptor belonging to MT(1) family (the binding is saturable, reversible, and inhibited by GTP analogs), and gives support for the presence of a functional melatonin receptor in the heart of the tench. In contrast, kinetic and pharmacological studies in the kidney revealed the preponderance of a melatonin binding site belonging to the MT(3)-like receptor subtype. Moreover, the decrease of specific binding in both, heart and kidney membranes, and the decrease of affinity in the kidney, produced by the addition of a non-hydrolysable GTP analog, and sodium cations suggest the presence of G(i/o)-proteins (that mediate inhibition of cAMP formation) coupled to such melatonin binding sites. Our results also point to different G(i/o)-proteins involved in the underlying mechanism of melatonin binding sites activation in the kidney. Additionally, the kinetics of [(125)I]Mel binding in kidney membrane preparations is a highly thermosensitive process, being necessary to perform the assays at 4 °C since the equilibrium was not reached at 25 °C assay temperature. The time needed to complete association of [(125)I]Mel at such low temperature is only 15s, whereas 100s is required to displace [(125)I]Mel specific binding by the unlabeled melatonin in kidney membranes. Present results support previous reports on melatonin effects in the regulation of different physiological functions in teleost (as cardiovascular physiology and osmoregulation) acting through peripheral specific receptors.

Burkholderia phymatum strains capable of nodulating Phaseolus vulgaris are present in Moroccan soils.

Phylogenetic analysis of 16S rRNA, nodC, and nifH genes of four bacterial strains isolated from root nodules of Phaseolus vulgaris grown in Morocco soils were identified as Burkholderia phymatum. All four strains formed N(2)-fixing nodules on P. vulgaris and Mimosa, Acacia, and Prosopis species and reduced acetylene to ethylene when cultured ex planta.

Circadian clock genes of goldfish, Carassius auratus: cDNA cloning and rhythmic expression of period and cryptochrome transcripts in retina, liver, and gut.

Clock genes are known to be the molecular core of biological clocks of vertebrates. They are expressed not only in those tissues considered central pacemakers, but also in peripheral tissues. In the present study, partial cDNAs for 6 of the principal clock genes (Period 1-3 and Cryptochrome 1-3) were cloned from a teleost fish, the goldfish (Carassius auratus ). These genes showed high homology (approximately 90%) with the respective cDNAs of zebrafish (Danio rerio), the only other teleost from which clock genes have been cloned. The daily expression pattern of each gene in retina, gut, and liver of goldfish was investigated using quantitative RT-PCR and cosinor analysis. All clock genes analyzed in the retina showed circadian rhythmicity; however, only Per 2-3 and Cry 2-3 were rhythmic in goldfish liver and gut. The amplitude and phase of the expression in liver and gut were different from those found in goldfish retina. Such differences suggest that other cues, such as feeding time, may contribute to the entrainment of oscillators in goldfish liver and gut. Our results support the use of goldfish as a teleost model to investigate the location and functioning of the circadian oscillators.

Effects of water salinity on melatonin levels in plasma and peripheral tissues and on melatonin binding sites in European sea bass (Dicentrarchus labrax).

Sea bass is an euryhaline fish that lives in a wide range of salinities and migrates seasonally from lagoons to the open sea. However, to date, the influence of water salinity on sea bass melatonin levels has not been reported. Here, we evaluated the differences in plasma and tissue melatonin contents and melatonin binding sites in sea bass under four different salinity levels: seawater (36 per thousand), isotonic water (15 per thousand), brackish water (4 per thousand) and freshwater (0 per thousand). The melatonin content was evaluated in plasma, whole brain, gills, intestine and kidney, while melatonin binding sites were analyzed in different brain regions and in the neural retina. Plasma melatonin levels at mid-dark varied, the lowest value occurring in seawater (102 pg/mL), and the highest in freshwater (151 pg/mL). In gills and intestine, however, the highest melatonin values were found in the seawater group (209 and 627 pg/g tissue, respectively). Melatonin binding sites in the brain also varied with salinity, with the highest density observed at the lower salinities in the optic tectum, cerebellum and hypothalamus (30.3, 13.0, and 8.0 fmol/mg protein, respectively). Melatonin binding sites in the retina showed a similar pattern, with the highest values being observed in freshwater. Taken together, these results reveal that salinity influences melatonin production and modifies the density of binding sites, which suggests that this hormone could play a role in timing seasonal events in sea bass, including those linked to fish migration between waters of different salinities for reproduction and spawning.

Free D-amino acids determination in ready-to-eat cooked ham irradiated with electron-beam by indirect chiral HPLC.

Potential racemization of L-amino acids (AA) in ready-to-eat (RTE) cooked ham after hygienization by electron-beam irradiation between 1 and 8kGy was studied. An indirect chiral method based on the derivatization reaction of AA with o-phthaldialdehyde and N-acetyl-L-cysteine followed by reversed-phase HPLC and fluorimetric detection was applied to detect ten enantiomeric pairs of free AA (Asp, Ser, Thr, Ala, Tyr, Val, Trp, Phe and Leu). Five of the D-AA were not found in any of the samples analyzed; the other five remaining D-AA (D-Asp, D-Ser, D-Ala, D-Val and D-Leu) were detected both in irradiated and non-irradiated cooked ham samples, their content being in the range 1.25-13.79µg/g. Although significant differences appeared for a few of the samples and doses, no positive correlation between the D-AA content and the irradiation doses was observed. Therefore, the electron-beam irradiation technique could be useful for sanitation of packed RTE cooked ham at doses allowed by WHO and EU, since it remains chemically safe to eat.

Choroidal neovascular membrane associated with unilateral retinitis pigmentosa. / Membrana neovascular coroidea asociada a retinitis pigmentosa unilateral.

A 67 year-old woman with diabetes mellitus type 2 no medical background of interest was attended in hospital due to visual loss of left eye of 4 months of onset. The fundus examination revealed findings corresponding to moderate non-proliferative diabetic retinopathy in the right eye and pigmented lesions similar to bone spicules and atrophy of the retinal pigment epithelium in the middle periphery and in the macular area in the left eye. The full-field electroretinogram was flat, with a slight insinuation of the b-wave in the light adaptation with a single flash of 3.0cd in the left eye. The optical coherence tomography showed the atrophic retina in all its layers, as well as intraretinal cysts and a serous neurosensory detachment of the macular retina with a lesion of high reflectivity in the left eye. Infectious and inflammatory diseases were ruled out. Three doses of intravitreal ranibizumab were administered monthly. The presence of choroidal neovascular membrane associated with unilateral retinitis pigmentosa has not been previously reported. The patient improved with intravitreal ranibizumab.

Ocular albinism with mutation in GPR143: Findings in wide-field autofluorescence and optical coherence tomography. / Albinismo ocular con mutación en GPR143: hallazgos en autofluorescencia de campo amplio y tomografía de coherencia óptica.

A 12 year-old boy who consulted due to nystagmus and low vision from birth. His mother also consulted for low vision of the right eye since she was a child, which worsened recently. The physical examination revealed no alterations in skin and hair pigmentation. In the examination of the anterior segment of the child, areas of slight circumferential hypopigmentation were observed in the iris in both eyes. The fundus examination revealed a choroidal fundus due to the absence of melanin in the retinal pigment epithelium. In the autofluorescence, an absence of physiological macular hypo-autofluorescence was observed and, in optical coherence tomography, foveal hypoplasia was observed in both eyes. In the ocular fundus examination of the mother, slight macular pigmentary changes were observed in the right eye, with hyperpigmented radiated spots in the retinal periphery of both eyes, which were hypo-autofluorescent in the wide-field autofluorescence. In the optical coherence tomography of the right eye, a cavitation of the outer retinal layers was observed in the fovea. The genetic study by nucleotide sequencing was performed on the mother and the child. In the mutation found in the GPR143 gene, the son was hemizygous and the mother was heterozygous. X-linked ocular albinism was diagnosed and the genetic counselling was carried out. Ocular albinism linked to X is the most frequent genetic variant of this disease. Peripheral pigment alterations in heterozygous mothers have been previously described in the literature, but there are no reports of cavitations in the external retinal layers using optical coherence tomography.

Molecular characterization of calcitonin gene-related peptide (CGRP) related peptides (CGRP, amylin, adrenomedullin and adrenomedullin-2/intermedin) in goldfish (Carassius auratus): cloning and distribution.

To further characterize the structure and function of calcitonin gene-related peptide (CGRP) related peptides in fish, we have cloned cDNA sequences for CGRP, amylin, adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) in goldfish (Carassius auratus) and examined their tissue distribution. CGRP, amylin, AM and IMD cDNAs were isolated by reverse transcription (RT) and rapid amplification of cDNA ends (RACE). The cloned sequences contain the complete four mature peptides, which present a high degree of identity with mature peptide sequences from other fish. Phylogenetic analyses show that goldfish AM and IMD form a sub-family within the CGRP-related peptides that is distinct from the CGRP/amylin sub-family. The distribution of goldfish CGRP-like peptides mRNA expression in different tissues and within the brain was studied by RT-PCR. CGRP, IMD and AM are detected throughout the brain, in pituitary and in most peripheral tissues examined. Amylin mRNA is mostly expressed in the brain, in particular posterior brain, optic tectum and hypothalamus, but is also present in pituitary, gonad, kidney and muscle. Our results suggest that goldfish CGRP, amylin, AM and IMD are conserved peptides that show the typical structure characteristics present in their mammalian counterparts. The widespread distributions of CGRP, AM and IMD suggest that these peptides could be involved in the regulation of many diverse physiological functions in fish. Amylin mRNA distribution suggests possible new roles for this peptide in teleosts, including the control of reproduction.

Determination of carbamate, phenylurea and phenoxy acid herbicide residues by gas chromatography after potassium tert-butoxide/dimethyl sulphoxide/ethyl iodide derivatization reaction.

The usefulness of the potassium tert-butoxide/dimethyl sulphoxide/ethyl iodide reaction with carbamate and phenylurea herbicides, and its application to phenoxy acids as a way to prevent hazards and toxicity of the sodium hydride/dimethyl sulphoxide/methyl iodide reaction was studied. Using factorial design optimization of this reaction was carried out. A solid-phase extraction method using dimethyl sulphoxide as eluent on-line with this reaction was developed to determine these herbicides in water samples by gas chromatography-mass spectrometry. Relative standard deviation values were lower than 10% for most of the herbicides in multicomponent trace determinations. Detection limits were in the 0.110-0.652 ng L(-1) concentration range. The validity of the method was confirmed by recovery studies from natural water samples.

Feeding entrainment of locomotor activity rhythms, digestive enzymes and neuroendocrine factors in goldfish.

The existence of food anticipatory activity (FAA) in animals subjected to daily feeding schedules seems to be mediated by a feeding-entrainable oscillator (FEO). Such an FEO may help in anticipating meal time and so optimizing food acquisition and nutrient utilization. In this study we investigated the existence of FAA and whether digestive enzymes, plasma cortisol, hypothalamic NPY and gastrointestinal tract (GIT) and plasma melatonin were entrained by periodic feeding in goldfish. We observed that periodically fed goldfish showed FAA in locomotor activity as well as in amylase and NPY. Alkaline protease and GIT melatonin were higher after feeding, whereas plasma cortisol levels were reduced. Plasma melatonin remained unmodified before and after meal time. These results suggested that scheduled feeding entrained both behavioral and certain physiological patterns in goldfish, FAA being of adaptive value to anticipate a meal and prepare the digestive physiology of fish.

Acute and chronic leptin reduces food intake and body weight in goldfish (Carassius auratus).

The purpose of the present study was to elucidate the possible role of leptin in food intake and body weight regulation in goldfish. We examined the effects of i.c.v. or i.p. acute leptin administration on food intake in food-deprived goldfish at different time intervals post-injection (0-2, 2-8 and 0-8 h). Food intake was reduced by i.p. administered leptin (1 microg) at 8 h post-injection, without statistically significant differences after i.c.v. treatment. The present study shows for the first time in a teleost that chronic (10 days) leptin treatment (i.p.) reduces food intake, body weight gain, specific growth rate and food efficiency ratio. Moreover, lipid and carbohydrate metabolism seems to be regulated by leptin in fish. Chronic leptin treatment increased lipid mobilization and carbohydrate storage as hepatic and muscle glycogen. Finally, leptin could mediate its actions on energy homeostasis in fish, at least in part, through interactions with hypothalamic catecholamines, since chronic leptin treatment reduced both hypothalamic noradrenergic and dopaminergic turnover without significant modifications in hypothalamic serotoninergic and neuropeptide Y (NPY) systems. In summary, our results suggest that leptin can regulate feeding behaviour and body weight homeostasis in fish.

Reassessment of the methyl derivatization reaction of carbamates with sodium hydride/dimethyl sulfoxide/methyl iodide for their determination by gas chromatography.

A deep revision of the carbamate methyl derivatization reaction with sodium hydride/dimethyl sulfoxide/methyl iodide was carried out. Representative carbamates, R(1)-NH-COO-R(2), mainly N-methyl and N-aryl ones, have been studied in order to clarify which carbamates undergo this reaction. Two possible reaction routes are proposed; the route depends on the carbamate substituent (-OR(2) group) more than on the methyl or aryl groups joined to the NH moiety as literature indicates. The classification of carbamates in N-methyl and N-aryl is not suitable to predict the methylation pathway. A laboratory-made closed reactor allows handling the reagents involved, minimizing hazards and simplifying the procedure for rapid analysis.

Indirect chiral HPLC determination and fluorimetric detection of D-amino acids in milk and oyster samples.

The indirect chiral method, based on the o-phthaldialdehyde reaction using the chiral N-acetyl-L-cysteine reagent, has been optimized to determine D-amino acids in milk and oyster samples. Both the derivatization reaction and the HPLC separation have been improved, and simple sample treatments were proposed. The milk sample preparation involved centrifugation and filtration through polytetrafluoroethylene filters for determination of free D-AA. Two methods, nonenzymatic and enzymatic, were applied to determination of free and total D-AA, respectively, in oyster samples. The D-AA contents were in the range of 0.14 to 4.32 mg/L for milk samples and 0.80 to 15.9 mg/g for oyster samples, with relative standard deviations lower than 10% (n = 4), except for D-Tyr. In general, mean recoveries were greater than 90% with relative standard deviations lower than 9% (n = 4) at concentration levels from 0.055 to 0.426 mg/L for milk samples and 0.348 to 1.10 mg/ g for oyster samples.

Periprandial changes and effects of short- and long-term fasting on ghrelin, GOAT, and ghrelin receptors in goldfish (Carassius auratus).

The periprandial profile and effects of short- (7 days) and long-term (30 days) fasting on the ghrelinergic system were studied in goldfish (Carassius auratus). Plasma levels of acyl-ghrelin, desacyl-ghrelin, and ghrelin O-acyl transferase (GOAT) were analyzed by enzymoimmunoassays, and expression of preproghrelin, goat and growth hormone secretagogue receptors (ghs-r) was quantified by real-time PCR. Circulating levels of acyl-ghrelin and GOAT rise preprandially, supporting the role of acyl-ghrelin as a meal initiator in this teleost. Consistently, preproghrelin and ghs-r1a1 expression increases 1 h before feeding time in intestinal bulb, suggesting that this receptor subtype might be involved in the preprandial action of ghrelin in this tissue. Significant postfeeding variations are detected for preproghrelin in telencephalon, goat in telencephalon and hypothalamus, ghs-r1a1 in vagal lobe, ghs-r1a2 and ghs-r2a1 in hypothalamus and ghs-r2a2 in telencephalon and vagal lobe, especially in unfed fish. Short- and long-term fasting significantly increase preproghrelin expression in telencephalon and gut. Goat expression is upregulated by short-term fasting in telencephalon and hypothalamus, and by both short- and long-term fasting in gut. Expression of ghs-r increases by fasting in telencephalon, while an upregulation of type 2, but not type 1, receptors is observed in vagal lobe. In intestinal bulb, ghs-r1a2 transcripts increase after both short- and long-term fasting. These results show a high dependence of the ghrelinergic system on feeding and nutritional status in fish, and demonstrate for the first time a differential implication of the various components of this system suggesting different roles for the four ghrelinergic receptor subtypes.

Nitrous Oxide Metabolism in Nitrate-Reducing Bacteria: Physiology and Regulatory Mechanisms.

Nitrous oxide (N2O) is an important greenhouse gas (GHG) with substantial global warming potential and also contributes to ozone depletion through photochemical nitric oxide (NO) production in the stratosphere. The negative effects of N2O on climate and stratospheric ozone make N2O mitigation an international challenge. More than 60% of global N2O emissions are emitted from agricultural soils mainly due to the application of synthetic nitrogen-containing fertilizers. Thus, mitigation strategies must be developed which increase (or at least do not negatively impact) on agricultural efficiency whilst decrease the levels of N2O released. This aim is particularly important in the context of the ever expanding population and subsequent increased burden on the food chain. More than two-thirds of N2O emissions from soils can be attributed to bacterial and fungal denitrification and nitrification processes. In ammonia-oxidizing bacteria, N2O is formed through the oxidation of hydroxylamine to nitrite. In denitrifiers, nitrate is reduced to N2 via nitrite, NO and N2O production. In addition to denitrification, respiratory nitrate ammonification (also termed dissimilatory nitrate reduction to ammonium) is another important nitrate-reducing mechanism in soil, responsible for the loss of nitrate and production of N2O from reduction of NO that is formed as a by-product of the reduction process. This review will synthesize our current understanding of the environmental, regulatory and biochemical control of N2O emissions by nitrate-reducing bacteria and point to new solutions for agricultural GHG mitigation.

Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).