Overview and New Insights Into the Diversity, Evolution, Role, and Regulation of Kisspeptins and Their Receptors in Teleost Fish

In the last two decades, kisspeptin (Kiss) has been identified as an important player in the regulation of reproduction and other physiological functions in vertebrates, including several fish species. To date, two ligands (Kiss1, Kiss2) and three kisspeptin receptors (Kissr1, Kissr2, Kissr3) have been identified in teleosts, likely due to whole-genome duplication and loss of genes that occurred early in teleost evolution. Recent results in zebrafish and medaka mutants have challenged the notion that the kisspeptin system is essential for reproduction in fish, in marked contrast to the situation in mammals. In this context, this review focuses on the role of kisspeptins at three levels of the reproductive, brain-pituitary-gonadal (BPG) axis in fish. In addition, this review compiled information on factors controlling the Kiss/Kissr system, such as photoperiod, temperature, nutritional status, sex steroids, neuropeptides, and others. In this article, we summarize the available information on the molecular diversity and evolution, tissue expression and neuroanatomical distribution, functional significance, signaling pathways, and gene regulation of Kiss and Kissr in teleost fishes. Of particular note are recent advances in understanding flatfish kisspeptin systems, which require further study to reveal their structural and functional diversity.Fil: Wang, Bin. Chinese Academy Of Fishery Sciences; ChinaFil: Mechaly, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; ArgentinaFil: Somoza, Gustavo Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Control neuroendocrí de l'inici de la maduresa sexual en els vertebrats mitjançant la kisspeptina i el seu receptor, amb especial èmfasi pel que fa a la situació en els peixos teleostis

El recent descobriment de la kisspeptina (producte del gen KISS1), conjuntament amb el seu receptor (producte del gen KISS1R), i de la seva relació amb el control de l'inici de la maduresa sexual en tots els vertebrats, ha estat descrit com una de les troballes més rellevants de la biologia reproductiva en els últims anys. La kisspeptina té un efecte directe sobre l'hormona alliberadora de gonadotrofines, la qual desencadena una cascada hormonal que finalment acaba a les gònades amb la secreció dels esteroides sexuals, els quals provoquen la consegüent maduració de l'individu. En aquest treball s'explica el descobriment dels gens KISS1 i KISS1R, l'estructura d'aquests gens i dels seus productes proteics, i es fa referència als mecanismes endocrins responsables tant de la regulació endògena com dels possibles factors externs que afecten el sistema kisspeptina-KISS1R. També, i amb especial èmfasi en els peixos teleostis, es discuteixen les possibles causes de les diferències i similituds trobades en l'expressió cerebral del sistema kisspeptina-KISS1R. Finalment, s'avaluen les perspectives futures en el camp del control de la reproducció mitjançant la manipulació del sistema kisspeptina-KISS1R.The discovery of kisspeptin (the product of the KISS1 gene) together with its receptor (product of the KISS1R gene), and the relationship with the control of the onset of puberty in all vertebrates has been described as one the most important findings in reproductive biology in the last years. Kisspeptin stimulates gonadotrophin-releasing hormone (GnRH), initiating the hormonal cascade which ends in the gonads with the secretion of the sex steroids which, in turn, drive sexual maturation. This paper describes the origin and discovery of the KISS1 and KISS1R genes, the structure of these genes as well as their protein products. The endocrine mechanisms and the exogenous factors affecting the kisspeptin- KISS1R complex are also discussed. Finally, we describe the multiple functions in relationship with this system in all vertebrates and discuss the differences and similarities regarding the neural expression of kisspeptin-KISS1R complex in several teleost fishes. Finally, the future perspectives in the field of the control reproduction by the manipulation of the kisspeptin-KISS1R complex are discussed

Changing the Receptor Specificity of Anthrax Toxin

The actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo

A review of the biology and ecology of Artemia persimilis Piccinelli & Prosdocimi, 1968 (Crustacea: Anostraca), as basis for its management

The genus Artemia (brine shrimp) is a small cosmopolitan crustacean, which primarily inhabits hypersaline water bodies, such as inland salt lakes, ponds and coastal lagoons. In Argentina two bisexual populations are encountered: Artemia franciscana and A. persimilis. The second is believed to be endemic to Argentina, but recently there have been some reports of their presence in a few locations in southern Chile. Artemia have been extensively studied because it is the most useful living food resource for the larval states of fish and crustaceans, and because of their unique reproductive strategies. Many authors in Argentina have agreed to indicate that A. persimilis needs special attention, and should be evaluated as a natural resource for aquaculture. The present paper provides a brief review of ecological aspects of A. persimilis and an overview of their use in aquaculture.Fil: Mechaly, Alejandro. University of Sidney; Australia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Angeletti, Sabrina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: De los Ríos Escalante, Patricio. Universidad Católica de Temuco; ChileFil: Cervellini, Patricia Marta. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Condiciones físicas del salitral de la vidriera y su relación con el zooplancton

El objetivo del presente trabajo es estudiar condiciones físicas y ambientales en el Salitral de la Vidriera, Provincia de Buenos. Aires, localizado en la zona interna del estuario de Bahía Blanca y su relación con el principal componente del zooplancton, el crustáceo Artemia persimilis (Crustacea, Anostraca). El enfoque se centró en la colecta del material biológico durante los meses de mayo a agosto de 2001. Los muéstreos se realizaron en dos transectas con tres estaciones cada una. Se midió temperatura, salinidad, pH y profundidad. Adicionalmente se registraron datos sobre llora y fauna acompañante y se realizó el análisis del tipo de sustrato. El zooplancton del Salitral de la Vidriera estuvo representado casi en su totalidad por Artemia persimilis. Los resultados indicaron que los meses de mayo y junio no difirieron estadísticamente en el número de individuos (73.66 y 57.34 ind/1 Olitros), pero si respecto al mes de agosto que registró una menor abundancia de 24.17 ind/101itros. Esta última variación podría estar condicionada a los cambios de salinidad y temperatura que fluctuaron durante ese mes en el cuerpo de agua. La temperatura del agua siguió el ritmo de la temperatura del aire, no registrándose fluctuaciones de magnitud importante en las diferentes transectas y en los distintos meses, registrándose temperaturas entre 12 - 15,5 °C en ese período. La salinidad fue muy baja (23.73), si se lo compara con el valor de 95.5 obtenido para diciembre de 2001. Este último registro sería más representativo para un ambiente hipersalino como el Salitral de la Vidriera. Los bajos valores de este parámetro estuvieron relacionados con una abundante precipitación para esos meses. El pH del agua fluctuó entre 7-8 y la profundidad en las dos transectas entre 25 y 55 cm. El estudio de las poblaciones evidenció un comportamiento bisexual, no detectándose que la relación machos/hembras fuera 1:1, pero si observándose una ligera superioridad de las hembras. Tampoco se registraron interacciones entre fechas y sectores, hallándose ambos sexos, a diferentes profundidades. En cuanto a las tallas se obtuvo un rango de ejemplares que variaron entre 5,88 - 8,98 mm. El 31.7 % de los individuos correspondió a adultos (talla > 5,88 mm).The objetive of this study was to analysed physical and environmental aspects of El Salitral de la Vidriera, Buenos Aires province and its relation with Artemia persimilis (Crustacea, Anostraca) one of the most important component of the zooplankton. Samples were taken during four months in winter time. Data of temperature, salinity, depth and pH were registered. Flora, fauna and substrate were analyzed. The results showed that the months of May and June did not differ statistically in the number of individuals, presenting values of 73.66 and 57.34 ind/1 Olitres but if differ from August, which registered an abundance of 24.17 ind/1 Olitres. This variation could be explain by changes in salinity and temperature, that fluctuated during that month. Water temperature varied 12 °C and 15,5 °C during the study period. Salinity was very low (23.73) comparig with the higher measurement (95.5) in december 2001. The last data is representative of the hypersaline environment of El Salitral de la Vidriera. The lowest values were related with the rainfall in preview months. The pH variation were 7-8 and depth between 25 to 55 cm. Populations showed a bisexual behavior, the relation male females not detected 1:1. A hight superiority of the females is observed. Interactions between dates and sectors were not registered. Both sexes, in different depths were found. Sizes of strain varied among 5,88 - 8,98 mm. The 31.7 % of the individuals were adults (size> 5,88 mm).Asociación Argentina de Geofísicos y Geodesta

Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors.Fil: Trajtenberg, Felipe. Instituto Pasteur de Montevideo; UruguayFil: Albanesi, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Ruetalo, Natalia. Instituto Pasteur de Montevideo; UruguayFil: Botti, Horacio. Instituto Pasteur de Montevideo; UruguayFil: Mechaly, Ariel. Instituto Pasteur de Montevideo; Uruguay. Institut Pasteur, Unité de Microbiologie Structurale, Paris; FranciaFil: Nieves, Marcos. Instituto Pasteur de Montevideo, Laboratorio de Biología Celular de Membranas, Montevideo; UruguayFil: Aguilar, Pablo Sebastián. Instituto Pasteur de Montevideo, Laboratorio de Biología Celular de Membranas, Montevideo; UruguayFil: Cybulski, Larisa Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Larrieux, Nicole. Instituto Pasteur de Montevideo; UruguayFil: de Mendoza, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; Uruguay. Institut Pasteur, Département de Biologie Structurale et Chimie, Paris; Franci

Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax

Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey

Morphology and Nanomechanics of Sensory Neurons Growth Cones following Peripheral Nerve Injury

A prior peripheral nerve injury in vivo, promotes a rapid elongated mode of sensory neurons neurite regrowth in vitro. This in vitro model of conditioned axotomy allows analysis of the cellular and molecular mechanisms leading to an improved neurite re-growth. Our differential interference contrast microscopy and immunocytochemistry results show that conditioned axotomy, induced by sciatic nerve injury, did not increase somatic size of adult lumbar sensory neurons from mice dorsal root ganglia sensory neurons but promoted the appearance of larger neurites and growth cones. Using atomic force microscopy on live neurons, we investigated whether membrane mechanical properties of growth cones of axotomized neurons were modified following sciatic nerve injury. Our data revealed that neurons having a regenerative growth were characterized by softer growth cones, compared to control neurons. The increase of the growth cone membrane elasticity suggests a modification in the ratio and the inner framework of the main structural proteins

Revisiting the pH-gated conformational switch on the activities of HisKA-family histidine kinases

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH