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1.
Proc Natl Acad Sci U S A ; 117(10): 5376-5385, 2020 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-32098846

RESUMEN

The mannose-6-phosphate isomerase (Mpi) locus in Semibalanus balanoides has been studied as a candidate gene for balancing selection for more than two decades. Previous work has shown that Mpi allozyme genotypes (fast and slow) have different frequencies across Atlantic intertidal zones due to selection on postsettlement survival (i.e., allele zonation). We present the complete gene sequence of the Mpi locus and quantify nucleotide polymorphism in S. balanoides, as well as divergence to its sister taxon Semibalanus cariosus We show that the slow allozyme contains a derived charge-altering amino acid polymorphism, and both allozyme classes correspond to two haplogroups with multiple internal haplotypes. The locus shows several footprints of balancing selection around the fast/slow site: an enrichment of positive Tajima's D for nonsynonymous mutations, an excess of polymorphism, and a spike in the levels of silent polymorphism relative to silent divergence, as well as a site frequency spectrum enriched for midfrequency mutations. We observe other departures from neutrality across the locus in both coding and noncoding regions. These include a nonsynonymous trans-species polymorphism and a recent mutation under selection within the fast haplogroup. The latter suggests ongoing allelic replacement of functionally relevant amino acid variants. Moreover, predicted models of Mpi protein structure provide insight into the functional significance of the putatively selected amino acid polymorphisms. While footprints of selection are widespread across the range of S. balanoides, our data show that intertidal zonation patterns are variable across both spatial and temporal scales. These data provide further evidence for heterogeneous selection on Mpi.


Asunto(s)
Manosa-6-Fosfato Isomerasa/genética , Selección Genética , Thoracica/enzimología , Thoracica/genética , Alelos , Animales , Sitios Genéticos , Genotipo , Isoenzimas/química , Isoenzimas/genética , Manosa-6-Fosfato Isomerasa/química , Mutación , Polimorfismo Genético
2.
Int J Biol Macromol ; 118(Pt A): 189-194, 2018 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-29890248

RESUMEN

Hydroxynitrile lyases (HNLs) are widely used in the asymmetric synthesis of cyanohydrins which are organic compounds used in the production of fine chemicals and pharmaceuticals, because these enzymes exhibit high catalytic efficiency and are very economical. In the present study, seeds of A. pedunculata Pall were identified as new potential source of HNLs. The HNL from A. pedunculata Pall (APHNL) was purified 138 fold and 4.20% yield with a specific activity of 661 U/mg. SDS-PAGE result showed the enzyme to be present as a monomer and the relative molecular mass determined by MALDI-TOF MS was 61 kDa. APHNL owned highest activity at pH 6.0 and at 60 °C temperature, showing activity up to 80 °C and stable up to 60 °C. APHNL has a Km of 0.5 mM, Vmax of 665.9 µmol mg-1 min-1, Kcat of 676.5 s-1 and Kcat/Km of 1353 s-1 mM-1 using mandelonitrile as substrate. Syntheses of (R)-mandelonitrile and (R)-2-Hydroxy-2-(3-phenoxy-phenyl)-acetonitrile were carried out using APHNL and molar conversion of (R)-mandelonitrile and (R)-2-Hydroxy-2-(3-phenoxy-phenyl)-acetonitrile were 90% and 98% with 94% and 93% ee, respectively. These results indicated that APHNL was an excellent biocatalyst and has very high potential for synthesis of enantiopure cyanohydrins.


Asunto(s)
Aldehído-Liasas/química , Semillas/química , Thoracica/química , Aldehído-Liasas/aislamiento & purificación , Animales , Catálisis , Cinética , Semillas/enzimología , Estereoisomerismo , Thoracica/enzimología
3.
Mar Pollut Bull ; 127: 505-511, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29475690

RESUMEN

In the present study, we analyzed the influence of untreated sewage exposure on carbon (δ13C) and nitrogen (δ15N) isotopic composition and several biochemical responses in the barnacle Balanus glandula. The main objective was to evaluate whether changes in stable isotopes signature do reflect biochemical sub-lethal effects in a sewage influence gradient. Stable isotopes analysis showed differences in isotope signatures between close sewage influence and distant sites, being δ13C signatures stronger than that of δ15N. Regarding biochemical effects, although organisms close to the effluent would be clearly exposed to contaminants (increased GST activity) the oxidative stress would not be too evident (peroxidases and ACAP not affected). The most affected physiological aspect was the digestive one, reflected in increased alkaline proteases and lipases activities. A clear relation between δ15N and GST activity was found, showing to δ15N as an indicator of potential exposure to chemical contaminants.


Asunto(s)
Isótopos de Carbono/análisis , Exposición a Riesgos Ambientales/análisis , Isótopos de Nitrógeno/análisis , Aguas del Alcantarillado/análisis , Thoracica/efectos de los fármacos , Contaminantes Químicos del Agua/análisis , Animales , Argentina , Proteínas Bacterianas/metabolismo , Biomarcadores/metabolismo , Endopeptidasas/metabolismo , Exposición a Riesgos Ambientales/efectos adversos , Lipasa/metabolismo , Agua de Mar/química , Aguas del Alcantarillado/efectos adversos , Thoracica/química , Thoracica/enzimología , Contaminantes Químicos del Agua/toxicidad
4.
J Exp Zool B Mol Dev Evol ; 326(4): 237-49, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27245369

RESUMEN

Energy metabolism is a key process in larval settlement of barnacles, but the underlying molecular mechanisms remain ambiguous. Arginine kinase (AK) mainly participates in energy metabolism in invertebrates. So far, its roles in barnacles have not been studied. In the present study, we raised an antibody against AK from Amphibalanus amphitrite Darwin to characterize the roles of AK in the larval settlement process. Among the developmental stages, AK was highly expressed during the cypris stage. Along with the aging process in cyprids, the level of AK decreased. The immunostaining results showed that AK was localized to muscular tissues in cyprids, including antennules, antennular muscles, and thoracic limbs. The larval settlement rate decreased and larval movement was inhibited in response to treatments with high concentrations of AK inhibitors (rutin and quercetin). These results demonstrated that AK was involved in the larval settlement of A. amphitrite through mediating energy supply in muscle tissues. Moreover, further analysis indicated that both the p38 MAPK and NO/cGMP pathways positively mediated the expression of AK in cyprids.


Asunto(s)
Thoracica/crecimiento & desarrollo , Animales , Arginina Quinasa/antagonistas & inhibidores , Arginina Quinasa/metabolismo , GMP Cíclico/metabolismo , Larva/enzimología , Larva/crecimiento & desarrollo , Músculos/enzimología , Quercetina/farmacología , Rutina/farmacología , Thoracica/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
5.
Sci Rep ; 5: 14767, 2015 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-26434953

RESUMEN

The MKK3-p38 MAPK pathway has been reported to mediate larval settlement in Amphibalanus (=Balanus) amphitrite. To clarify the underlying molecular mechanism, we applied label-free proteomics to analyze changes in the proteome of cyprids treated with a p38 MAPK inhibitor. The results showed that the expression levels of 80 proteins were significantly modified (p < 0.05). These differentially expressed proteins were assigned to 15 functional groups according to the KOG database and 9 pathways were significantly enriched. Further analysis revealed that p38 MAPK might regulate the energy supply and metamorphosis. Two potential regulatory proteins, CUB-serine protease and PKAα, were both down-regulated in expression. CUB-serine protease localized to postaxial seta 2 and 3, as well as the 4 subterminal sensilla in the antennule. Importantly, it was co-localized with the neuron transmitter serotonin in the sections, suggesting that the CUB-serine protease was present in the neural system. PKAα was highly expressed during the cyprid and juvenile stages, and it was co-localized with phospho-p38 MAPK (pp38 MAPK) to the cement gland, suggesting that PKAα might have some functions in cement glands. Overall, p38 MAPK might regulate multiple functions in A. amphitrite cyprids, including the energy supply, metamorphosis, neural system and cement glands.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Thoracica/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Secuencia Conservada , Retroalimentación Fisiológica , Imidazoles/farmacología , Larva/efectos de los fármacos , Larva/enzimología , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Especificidad de Órganos , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , Proteoma/metabolismo , Piridinas/farmacología , Serina Endopeptidasas/metabolismo , Thoracica/efectos de los fármacos
6.
Toxins (Basel) ; 7(8): 2739-56, 2015 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-26213967

RESUMEN

Effective and ecofriendly antifouling (AF) compounds have been arising from naturally produced chemicals. The objective of this study is to use cyanobacteria-derived agents to investigate the role of acetylcholinesterase (AChE) activity as an effect and/or mode of action of promising AF compounds, since AChE inhibitors were found to inhibit invertebrate larval settlement. To pursue this objective, in vitro quantification of AChE activity under the effect of several cyanobacterial strain extracts as potential AF agents was performed along with in vivo AF (anti-settlement) screening tests. Pre-characterization of different cholinesterases (ChEs) forms present in selected tissues of important biofouling species was performed to confirm the predominance of AChE, and an in vitro AF test using pure AChE activity was developed. Eighteen cyanobacteria strains were tested as source of potential AF and AChE inhibitor agents. Results showed effectiveness in selecting promising eco-friendly AF agents, allowing the understanding of the AF biochemical mode of action induced by different compounds. This study also highlights the potential of cyanobacteria as source of AF agents towards invertebrate macrofouling species.


Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Cianobacterias/metabolismo , Mytilus/enzimología , Thoracica/enzimología , Animales , Bencenamina, 4,4'-(3-oxo-1,5-pentanodiil)bis(N,N-dimetil-N-2-propenil-), Dibromuro/farmacología , Incrustaciones Biológicas , Cobre/farmacología , Mytilus/efectos de los fármacos , Fisostigmina/farmacología , Tetraisopropilpirofosfamida/farmacología , Thoracica/efectos de los fármacos
7.
J Exp Biol ; 218(Pt 16): 2505-9, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26113139

RESUMEN

RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in 'double transfections', in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments.


Asunto(s)
ARN Interferente Pequeño/genética , Thoracica/genética , Animales , Larva/enzimología , Larva/genética , Metamorfosis Biológica , Interferencia de ARN , Thoracica/enzimología , Thoracica/crecimiento & desarrollo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/genética
8.
PLoS One ; 8(10): e77069, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24130836

RESUMEN

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO2 levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.


Asunto(s)
Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Thoracica/enzimología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Exones/genética , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Salinidad , Agua de Mar/química , Alineación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/genética , Thoracica/crecimiento & desarrollo , Thoracica/fisiología
9.
Neurosci Lett ; 555: 209-14, 2013 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-24076140

RESUMEN

The Amphibalanus amphitrite barnacle is a sessile marine crustacean and a major constituent of benthic as well as intertidal communities. A. amphitrite is also an important component of biofouling on artificial substrates. The role of nitric oxide (NO) was recently observed in the settlement of this species. In this work, we used immunohistochemical and histoenzymatic methods to investigate, for the first time, the presence and distribution of NO synthetic enzymes (NOS) in the competent-for-settlement cyprid of A. amphitrite. NOS-like immunoreactivity was observed in various regions of the cyprid: gut mucosa, mantel epithelium, thoracic muscle, and abductor muscles. Intense immunoreactivity was also present in the cement gland and oil cells, while widespread immunoreactivity was observed in the compound eye. NADPH-diaphorase method was used to provide further data and understand NOS-distribution. The results show that NOS is likely to be present in structures - such as muscles and cement gland - which are key for settlement.


Asunto(s)
Óxido Nítrico Sintasa/metabolismo , Thoracica/enzimología , Animales , NADPH Deshidrogenasa/metabolismo , Especificidad de Órganos
10.
PLoS One ; 8(7): e69510, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23922727

RESUMEN

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.


Asunto(s)
Larva/enzimología , Larva/crecimiento & desarrollo , MAP Quinasa Quinasa 3/metabolismo , Thoracica/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , MAP Quinasa Quinasa 7/genética , MAP Quinasa Quinasa 7/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética
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