Monogenoideans (Platyhelminthes) from the gill lamellae of the spotted sea trout, Cynoscion nebulosus (Perciformes, Sciaenidae), from the western coast of the Yucatan Peninsula, Mexico, with redescription of Diplectanum bilobatus Hargis 1955 (Diplectanidae).

The gill lamellae ectoparasites of the spotted sea trout, Cynoscion nebulosus (Sciaenidae), in the western coast of the Yucatán Peninsula, Mexico, revealed three species of Monogenoidea: Cynoscionicola heteracantha (Manter 1938) Price, 1962 (Microcotylidae); Choricotyle cynoscioni (MacCallum, 1917) Llewellyn, 1941 (Diclidophoridae); and Diplectanum bilobatus Hargis, 1955 (Diplectanidae). Brief comments about the current taxonomic status as well as supplemental observations of all these monogenoids originally described and/or reported from the same host fish species found in the USA are provided. New illustrations, prevalence and mean intensity of infection, as well morphological and biometric data based on new specimens are shown. C. heteracantha and C. cynoscioni collected in this study represent the second and first records of the species of these genera for the Atlantic coast of Mexico. The specimens of D. bilobatus are provisionally retained within Diplectanum until an emendation of the genus and a formal revision of all named species of this monogenoidean genus are undertaken.

Cocaine modulates the expression of transcription factors related to the dopaminergic system in zebrafish.

Nodal-related protein, Ndr2, and transcription factors such as Lmx1b, Otp, Nurr1 and Pitx3 are very important in the differentiation, function and maintenance of mesodiencephalic dopaminergic neurons, and are necessary for the activation of tyrosine hydroxylase (TH) and dopamine (DA) transporter expression. Hence, the aim of the present work was to evaluate the effects of cocaine on the expression of genes related to the embryogenesis development of the dopaminergic system. Zebrafish embryos were exposed to cocaine hydrochloride at 5h post-fertilization (hpf), and collected at two important stages - 24 and 48hpf - to study the effects of cocaine on the expression of ndr2, the lmx1b.1, lmx1b.2, otpa, otpb, nurr1 transcription factors, and their target genes: TH and DA transporter expression. Our results by qPCR showed that cocaine affects the expression of these genes in different ways, depending on the stage of development. Furthermore by in situ hybridization we observed a change in the spatial distribution of lmx1b.1 and lmx1b.2 at both stages (24 and 48hpf) due to exposure to cocaine. We also show the importance of Lmx1b and Otp in th expression through the knockdown of Lmx1b.1 and Lmx1b.2, and of Otpa and Otpb. Additionally, cocaine produced an increase and a decrease in TH levels at 24 and at 48hpf, respectively, possibly due to the change in the expression of the transcription factors and ndr2 expression. We conclude that cocaine alters the correct development of dopaminergic system affecting the expression of transcription factors, during the embryogenesis.

New insights into opioid regulatory pathways: influence of opioids on Wnt1 expression in zebrafish embryos.

Opioids are the most potent analgesics known today, but their prolonged administration produces severe adverse effects such as constipation, bradycardia, besides addiction, a concept not fully understood at present, which represents one of the most important challenges of modern bioscience. Wnts constitute an important family of vertebrate genes that encode secreted signaling proteins implicated in various developmental processes (patterning of the neural tube, neuronal differentiation), and are extensively conserved through evolution. In this study we have focused on Wnt1, an essential signal in axis polarity, as well as in proliferation and the development and differentiation of the CNS, roles shared by opioid receptors. Our previous studies in zebrafish show that morphine, the most potent analgesic known today, increases cell proliferation and induces neuronal protection and dopaminergic differentiation by activating the opioid receptors. The aim of the present study is to determine whether these effects are a consequence of an interaction between Wnt1 and the endogenous opioid system, which may act as a transcription regulator of Wnt1. Hence, we have exposed embryos to morphine, the endogenous delta opioid agonist Met-Enkephalin-Glu-Tyr (MEGY) (it binds with high affinity to both zebrafish delta opioid receptors, ZfDORs), and SNC80, a highly specific delta agonist, which displays low affinity towards the ZfDORs. Although at earlier stages, all opioids reduced the expression level of Wnt1, further on development, mainly during the differentiation of the CNS (24-48 h post fertilization (hpf)), morphine and MEGY increased Wnt1 expression. Our results point to the possibility that opioid signaling controls the transcription of Wnt1 and that through Wnt1, the opioid system regulates cell proliferation and neuronal differentiation. The present work opens a door to the discovery of new mechanisms that regulate opioid activity and its adverse effects, and hence, it might provide a good target to design new drugs that prevent or avoid these effects.

The validity of incremental exercise testing in discriminating of physiological profiles in elite runners.

The goal of this study was to determine whether traditional ergoespirometric incremental exercise testing carried out to the point of exhaustion could be useful in distinguishing the physiological profiles of elite runners that compete in races that lasted about 8 minutes versus those that lasted about 2 hours. Ten male marathon runners (performance time: 2:12:04, coefficient of variation (CV) = 2.33%) and 8 male 3000 m steeplechase runners (performance time: 8:37.83, CV = 2.12%) performed an incremental test on the treadmill (starting speed 10 km·h-1; increments, 2 km·h-1; increment duration, 3 min to exhaustion). Heart rate (HR), VO2, and lactate concentrations were measured at the end of each exercise level. At maximal effort, there were no differences between the groups regarding VO2max and maximal HR; however, the workload time, vVO2max and peak treadmill velocity were significantly higher in the 3000 m steeplechase group (p<0.05). At submaximal effort, there were no significant differences between groups for VO2 (ml·kg-1·min-1), HR, or lactate. Our results show that this type of testing was not sufficient for discriminating the physiological profiles of elite runners who competed in middle-distance versus long-distance events (e.g. in the marathon and the 3000 m steeplechase).

Expression of the nociceptin receptor during zebrafish development: influence of morphine and nociceptin.

The NOP system is considered to be part of the opioid system, although it exerts antiopioid actions depending on the anatomical region where it is localized. This apparent controversy has lead to the hypothesis that the NOP system interacts with the classical opioid systems (mu, delta, kappa) and regulates/modulates their activity in relation to analgesia and the development of addiction to drugs. In order to shed light into the importance of the NOP system, we have analyzed the expression of NOP during zebrafish development, and the effect of its endogenous agonist nociceptin and the opioid agonist morphine on NOP expression. Our qPCR study show that the number of NOP transcripts is different at each developmental stage studied (0.5 hpf, 2.75 hpf, 3 hpf, 8 hpf, 16 hpf, 19 hpf, 22 hpf, 24 hpf, 30 hpf, 48 hpf, 60 hpf and 72 hpf). Nociceptin enhances NOP expression at 24 hpf but decreases the number of NOP copies at 48 hpf, whereas NOP expression decreases after morphine exposure at 24 hpf and 48 hpf. Also, our ISH analysis demonstrates that nociceptin causes a change in the distribution of NOP towards rostral areas at both developmental stages. Morphine produces similar changes to those of nociceptin although only at 48 hpf. The present work leads to the conclusion that the NOP system is important during embryogenesis. Exposure to drugs changes the expression level and localization of NOP, suggesting that also during development, NOP plays a role in the apparition of dependence and addiction to drugs.

Developmental expression and distribution of opioid receptors in zebrafish.

Zebrafish is a novel experimental model that has been used in developmental studies as well as in the study of pathological processes involved in human diseases. It has been demonstrated that the endogenous opioid system is involved in developmental mechanisms. We have studied the relationship between the different embryonic stages and opioid receptor expression for the four known opioid receptors in zebrafish (mu, delta 1, delta 2 and kappa). The mu opioid receptor is detected at higher levels than the other opioid receptors before the midblastula transition and during the segmentation period. The delta duplicate 2 exhibits only one peak of expression at 21 h postfertilization (hpf), when the motor nervous system is forming. The kappa receptor is expressed at very low levels. In situ hybridization studies at 24 hpf show that the opioid receptors are widely distributed in zebrafish CNS and at 48 hpf their localization is detected in more defined structures. Our results support specific implications of the opioid receptors in developmental processes such as morphogenesis of the CNS, neurogenesis, neuroprotection and development of neuromuscular and digestive system. Pain-related alterations can be a consequence of changes in the endogenous opioid system during development, hence we provide important information that might help to solve pain-related pathological situations.

Characterization of two duplicate zebrafish Cb2-like cannabinoid receptors.

Several cannabinoid receptors have been detected in many organisms. The best known are CB1, mainly expressed in the central nervous system and CB2 which is almost exclusively expressed in the periphery. Here we report the molecular characterization of two duplicate CB2-like cannabinoid receptors from zebrafish (Danio rerio) (zebrafish Cb2a and zebrafish Cb2b). The amino acid sequences of these receptors present 56% identity with Takifugu rubripes CB2 sequence and 39% with human CB2 sequence and conserve some specific key residues for cannabinoid receptor function. Both duplicate receptors are expressed in peripheral tissues (gills, heart, intestine and muscle), immune tissue (spleen) and also in the central nervous system. Using in situ hybridization techniques zebrafish Cb2 mRNA expression was observed for the first time in the adenohypophysial cells of the rostral pars distalis and proximal pars distalis of the pituitary gland. Given the importance of the existence of duplication of genes in teleosts, the combined analysis of these two new cannabinoid receptors opens a new exciting door to investigate and understand cannabinoid function throughout evolution.

Identification of dynorphin a from zebrafish: a comparative study with mammalian dynorphin A.

We report the cloning and molecular characterization of the zfPDYN. The complete open reading frame for this propeptide is comprised in two exons that are localized on chromosome 23. zfPDYN cDNA codes for a polypeptide of 252 amino acids that contains the consensus sequences for four opioid peptides: an Ile-enkephalin, the neo-endorphins, dynorphin A and dynorphin B. Upon comparison between zebrafish (zfDYN A) and mammalian dynorphin A (mDYN A) it has been stated that these two peptides only differ in two amino acids: the Leu(5) is replaced by Met(5) and the Lys(13) by Arg(13). Taking into consideration that mDYN A is able to bind to the three mammalian opioid receptors, we have compared the pharmacological profile of zfDYN A and mDYN A on the zebrafish opioid receptors. By means of radioligand binding techniques, we have established that these two dynorphins bind and activate all of the cloned opioid receptors from zebrafish (delta-, mu- and kappa-like), although with different affinities. zfDYN A and mDYN A displace [(3)H]-diprenorphine binding with K(i) values on the nanomolar range, showing greater affinity for zebrafish opioid receptor (ZFOR) 3 (kappa) receptor. ZFOR1 (delta) and ZFOR4 (delta) present higher affinity for zfDYN A than for mDYN A, while the opposing behavior is observed in ZFOR2 (mu). Functional [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) stimulation experiments indicate that these two peptides fully activate the zebrafish opioid receptors, although the mean effective dose (EC(50)) values obtained for ZFOR2 and ZFOR3 receptors are lower than those seen for ZFOR1 and ZFOR4. A comparative study indicates that mammalian and zebrafish opioid receptors might bind their corresponding dynorphin A in a similar fashion, hence suggesting an important role of the opioid system through the vertebrate evolution.

Binding profile of the endogenous novel heptapeptide Met-enkephalin-Gly-tyr in zebrafish and rat brain.

Zebrafish is considered a model organism, not only for the study of the biological functions of vertebrates but also as a tool to analyze the effects of some drugs or toxic agents. Five opioid precursor genes homologous to the mammalian opioid propeptide genes have recently been identified; one of these, the zebrafish proenkephalin, codes a novel heptapeptide, the Met-enkephalin-Gly-Tyr (MEGY). To analyze the pharmacological properties of this novel ligand, we have labeled it with tritium ([(3)H]MEGY). In addition, we have also synthesized two analogs: (d-Ala(2))-MEGY (Y-d-Ala-GFMGY) and (d-Ala(2), Val(5))-MEGY (Y-d-Ala-GFVGY). The binding profile of these three agents has been studied in zebrafish and rat brain membranes. [(3)H]MEGY presents one binding site in zebrafish, as well as in rat brain membranes, although it shows a slight higher affinity in zebrafish brain. The observed saturable binding is displaced by naloxone, thus confirming the opioid nature of the binding sites. Competition binding assays indicate that the methionine residue is essential for high-affinity binding of MEGY and probably of other peptidic agonists in zebrafish, whereas the change of a Gly for a d-Ala does not dramatically affect the ligand affinity. Our results show that the percentage of [(3)H]MEGY displaced by all the ligands studied is higher than 100%, thus inferring that naloxone (used to determine nonspecific binding) does not bind to all the sites labeled by [(3)H]MEGY. Therefore, we can deduct that some of the MEGY binding sites should not be considered classical opioid sites.

Expression of the mu-opioid receptor in the anterior pituitary gland is influenced by age and sex.

To analyze whether opioids are able to modulate endocrine regulation by acting directly on rat pituitary cells, an immunohistochemical study of micro-opioid receptor expression in these cells was performed, with attention given to the analysis of potential age- and sex-related variations in receptor expression patterns. In both sexes, the micro-opioid receptor was detected in the pituitary pars distalis. However, significant age-related differences were observed. Both in male and female rats, the percentage of micro-opioid receptor-expressing cells decreased significantly from postnatal week one through the 24 months of our study. Interestingly, pituitary cells containing the micro-opioid receptor were significantly more numerous in male than in female, with exception of the pre-pubertal phase and old rats. According to two-way analysis of variance, the gender-related differences in micro-receptor expression were independent of age-related variations. Thus, without excluding hypothalamic actions, our results suggest that opioids may exert their endocrine function by acting directly on pituitary cells.

Cloning and characterization of a full-length pronociceptin in zebrafish: evidence of the existence of two different nociceptin sequences in the same precursor.

We report the cloning and molecular characterization of a zebrafish pronociceptin gene, which is expressed in brain and intestine. This fish precursor codes for two different nociceptin peptides, one of which presents an opioid sequence in its N-terminus, being more similar to dynorphin A than its mammalian counterparts. Our results represent a new tool to investigate the function of nociceptin and its receptor in relation to pain and drug addiction.

Characterization of zebrafish proenkephalin reveals novel opioid sequences.

Cloning and molecular characterization of an homologous gene to proenkephalin in a teleost, the zebrafish Danio rerio is presented in this paper. The new zebrafish proenkephalin (zfPENK) encodes a 249 amino acid polypeptide that displays an identity of 40% to mammalian PENKs and which contains the consensus sequences for four Met-enkephalins, one Leu-enkephalin and one Met-enkephalin-Gly-Tyr, described for the first time in teleosts. Expression studies indicate that zfPENK is selectively expressed in zebrafish brain. Our findings support the concept that PENK genes might have been highly conserved throughout evolution and that its origins might be placed more than 400 million years ago. Moreover, we present evidence that the heptapeptide Met-enkephalin-Gly-Tyr present in fish might be anterior in evolution to the heptapeptide Met-enkephalin-Arg-Phe present in tetrapods. Also another homologous sequence to proenkephalin in zebrafish genome is presented. This sequence might stand for the third exon of a possible duplicate gene of zfPENK. Our findings not only present new data in relation to the evolution of opioid peptides in vertebrates, but also we present a new heptapeptide with putative differential activity than the other peptides derived from the mammalian proenkephalins. Future research will define the functional role of this new heptapeptide in the mechanism that describes opioid activity.

Growth hormone expression in ontogenic development in gilthead sea bream.

The pattern of expression of the growth hormone (GH) gene was studied during the early development of gilthead sea bream ( Sparus aurata). The GH transcript was detected from the 2nd day of the larval stage onwards. In the next stages the expression level fluctuated, possibly due to different regulatory factors. The distribution of GH mRNA studied by in situ hybridization (ISH) was found to be pituitary specific. Hybridization signals for GH mRNA were detected for the first time in 4-day-old larvae. Throughout development the cells that express GH mRNA were mainly located in the proximal pars distalis. Mammosomatotroph cells coexpressing GH and PRL were not detected in juveniles or adults. Moreover, the possible involvement of GH in asynchronic growth in cultivation of gilthead sea bream was also examined by ISH. No differences in the distribution of GH cells were observed in the three sizes of juveniles of gilthead sea bream studied. These results suggest that the transcription of GH is involved in the early developmental stages of sea bream and the asynchronous growth-related changes are not due to distinct distribution of GH cells.

Transient expression pattern of prolactin in Sparus aurata.

Using reverse transcription-polymerase chain reaction and in situ hybridization, the expression of the prolactin (PRL) gene was determined during development in gilthead sea bream (Sparus aurata) for the first time. The mRNA for PRL was detected from the second day of the larval stage onwards. This transcript was also located in the adenohypophysial cells, starting at four days post-hatching and was found to be pituitary-specific. Moreover, the possible involvement of PRL in asynchronous growth in the cultivation of gilthead sea bream was also examined. No differences in the distribution of PRL cells were observed in the three sizes of juvenile gilthead sea bream studied. These results suggest that the transcription of PRL is involved in the early development stages of sea bream and that the asynchronous growth-related changes are not due to distinct distribution of PRL cells.

[Analysis of the HFE gene in a large Spanish kindred with hereditary hemochromatosis]. / Estudio del gen HFE en una familia española con hemocromatosis hereditaria.

BACKGROUND: Hereditary hemochromatosis (HH) is an inborn error of iron metabolism that is inherited as an autosomal recessive trait. Recently, it has been shown that the HFE gene, located telomeric to HLA-A on chromosome 6 is mutated in most patients with HH. Moreover, the phenotypic expression of hereditary hemochromatosis is influenced by sex and environmental agents such as alcohol and dietary iron intake. SUBJECTS AND METHOD: We have studied 40 subjects from a family some of which members have HH. DNA was obtained from nucleated peripheral blood cells, and exons 2 and 4 and intron 5 of the HFE gene were amplified by PCR and digested with specific restriction enzymes. RESULTS: Analysis of the HFE gene revealed that 29 members of the family carry some of the three HFE mutations (C282Y, H63D and S65C). Nevertheless, only those homozygous for the C282Y mutation develop HH. In this family, the allele 187G of exon 2 mutation is cosegregated with the allele IVS5-47 A in intron 5. CONCLUSIONS: Analysis of the HFE gene in the members of a large Spanish kindred, living in the same geographical area, shows that only those homozygous for the C282Y mutation develop hemochromatosis.

ZFOR2, a new opioid receptor-like gene from the teleost zebrafish (Danio rerio).

A new opioid receptor-like (ZFOR2) has been cloned and characterized in an anamniote vertebrate, the teleost zebrafish (Danio rerio). ZFOR2 encodes a 384-amino-acid protein with seven potential transmembrane domains, and its predicted amino acid sequence presents an overall 74% degree of identity to mammalian mu opioid receptors. Its inclusion in a dendrogram generated from the alignment of the opioid receptor's protein sequences, confirms its classification as a mu opioid receptor. Divergences in sequence are greater in the regions corresponding to extracellular loops, suggesting possible differences in ligand selectivity with respect to the classical mu opioid receptors. The genomic structure of ZFOR2 is also highly conserved throughout the phylogenetic scale, supporting the origin of opioid receptors early in evolution. Nevertheless, ZFOR2 lacks the fourth exon found in human and rodent mu opioid receptors, that is known to be involved in desensibilization and internalization processes.

Polymorphism in the interleukin-1 receptor antagonist gene is associated with alcoholism in Spanish men.

BACKGROUND: A polymorphism located in intron 2 of the interleukin-1 receptor antagonist (IL1RN) gene recently has been associated with the development of hepatic fibrosis in Japanese alcoholics. In the present study, we analyzed whether there is an association between this polymorphism, alcoholism, and alcoholic liver disease in a Spanish male population of alcoholics. METHODS: The IL1RN genotype was assessed by polymerase chain reaction by using oligonucleotides that flank a variable nucleotide tandem repeat polymorphism located in intron 2 of this gene in 90 male alcoholic patients from Spain: 30 alcohol-dependent men, 30 alcohol abusers, and 30 alcoholics with liver cirrhosis. We also studied 40 healthy subjects. RESULTS: The distribution of the IL1RN allelic frequencies in Spanish healthy subjects is similar to that previously reported in White subjects. However, the A1 allele is overrepresented in Spanish alcoholics when compared with healthy subjects. No significant differences in allelic frequencies were observed between alcoholics with liver cirrhosis and alcoholics without liver disease or between alcohol-dependent subjects and alcohol abusers. CONCLUSION: The presence of the A1 allele of the IL1RN gene is associated with a higher risk of alcoholism in Spanish men.

Characterization of ZFOR1, a putative delta-opioid receptor from the teleost zebrafish (Danio rerio).

ZFOR1 is a putative opioid receptor from zebrafish brain which has 66% homology with the mammalian delta-opioid receptor. When expressed in HEK293 cells ZFOR1 bound the non-selective opioid antagonist [(3)H]diprenorphine with high affinity. However, the binding of this ligand was not readily displaced by opioids selective for mu, delta or kappa opioid receptors (affinities>1000 nM). Rather non-selective ligands showed good affinity, as did the non-peptide delta-ligand BW373U86 (Ki 69 nM), the delta-antagonist naltrindole (Ki 28 nM) and the peptide beta-endorphin (Ki 37 nM). Agonist binding to the receptor encoded by ZFOR1 receptor stimulated the binding of [(35)S]GTPgammaS confirming coupling to G proteins. Study of the receptor should contribute to understanding of the evolution of the opioid system.

Evaluation of hydrogen line emission and argon plasma electron concentrations resulting from the gaseous sample injection involved in hydride generation-ICP-atomic emission spectrometric analysis.

The simultaneous injection of volatile hydride species and hydrogen gas, originating in reagent decomposition, was monitored during the operation of a continuous hydride generation manifold employed for the determination of trace arsenic by HG-ICP-AES. Line and background intensities as well as the FWHM of the hydrogen Hgamma and Hdelta lines were measured, and electron number densities (ne) estimated from Stark broadening of the line profiles. Results were compared with those obtained by conventional pneumatic injection of aqueous solutions. Overlapping with atomic nitrogen lines at 410 nm and 411 nm tends to distort the Hdelta line profile for the hydrogen-seeded plasma, rendering unreliable results. The N I lines seem to be quenched by the presence of water aerosol. More consistent results were obtained with the Hgamma line. When no solutions are pumped through the hydride generation manifold ("dry" plasma), the measured ne value was (1.57 +/- 0.22) x 10(15)cm(-3). Conversely, when the reducing reagent flow was replaced by pure water (corresponding to the injection of water vapor in equilibrium that is swept by the argon carrier gas passing through the phase separator), the electron concentration is 25% higher. In that case the ne value agrees between the experimental error with that obtained for a plasma in which a water aerosol is introduced at a flow rate of 1 mL/min. An enhancement of 52% relative is observed in ne when the system is operated under optimized conditions for arsine generation, employing sodium tetrahydroborate in acidic medium as reducing agent (i.e. hydrogen seeded plasma). It was also observed that the continuum emission near 410 nm for the hydrogen containing plasma correlates with the measured electron number density, suggesting that the background enhancement under hydride generation conditions may respond to the ion-electron recombination mechanism.

The Micro-Reflux Test: a new test to evaluate nasolacrimal duct obstruction.

OBJECTIVE: The authors describe a new and simple procedure, the Micro-Reflux Test (MRT), to screen for primary acquired nasolacrimal duct obstruction (PANDO). This study sought to determine the reliability of this new test in the diagnosis of complete nasolacrimal duct obstruction. DESIGN: A nonrandomized, prospective, self-controlled, comparative case series. PARTICIPANTS: Two hundred eyes of 100 patients with documented unilateral complete PANDO were examined. INTERVENTION: The MRT was performed by a masked examiner on both eyes of each patient. Two drops of 0.25% sodium fluorescein dye were instilled in the inferior cul-de-sac and the patient made to blink five times to activate the lacrimal pump mechanism. Excess fluorescein dye was blotted away using tissue paper. The patient was positioned at the slit lamp, and observation of the inferior punctum with the cobalt blue filter was done using 5x magnification. The tissue overlying the lacrimal sac was massaged in a counterclockwise direction with moderate pressure using the index finger. The test was considered positive if there was continued observed reflux of fluorescein-stained tears from the inferior punctum after the initial counterclockwise massage to empty the inferior canaliculus. The validity of the MRT was measured by sensitivity and specificity as well as positive- and negative-predictive values. MAIN OUTCOME MEASURE: The MRT was considered positive if there was continued reflux of fluorescein-stained tears from the inferior punctum after the initial digital massage to empty the dye from the inferior canaliculus. RESULTS: The MRT used for the evaluation of complete PANDO has a sensitivity of 97% and a specificity of 95%. It has a positive-predictive value of 95% and a negative-predictive value of 97%. CONCLUSION: The MRT is a reliable screening test for presence of complete nasolacrimal duct obstruction.