Hermaphroditism, associated with territoriality and dominance behavior, is common in the marine environment. Male sex-specific coloration patterns and behavior are particularly evident in species where males are territorial and guard harems of females such as wrasses and groupers. Protogynous hermaphrodites that change sex from female to male are good models to study sexual behavior and related changes in the brain due to their abilities to reorganize their sexual phenotype as adults. Two hormones produced in the brain and implicated in the process of sex-specific behavior and reproductive development are arginine vasotocin (AVT) and gonadotropin releasing hormone (GnRH). While a wealth of data exists regarding these hormone systems separately, little is known about linkage between these two systems. Especially there is no data tracking these two systems together in any protogynous fish. This study was conducted to test the hypothesis that coordinated interactions between AVT and GnRH facilitate the process of behavioral and gonadal sex change in the rock hind Epinephelus adscensionis. Four topics were addressed to investigate the relationship between behavior and reproduction: i) rock hind sex change, sexual characteristics and conditions causing sex change to occur in captivity were detailed as a basis for examining the AVT system and GnRH during this process, ii) the distribution of a vasotocin V1a type receptor identified in rock hind brain was examined for the first time in a fish species using a custom designed antibody then the receptor protein was co-localized with GnRH producing cells within the brain to confirm that a pathway exists for AVT action on GnRH, iii) levels of AVT, AVT receptors, and GnRH messenger RNA (mRNA) were compared between male and female rock hind phenotypes, and iv) female rock hind at early stages of sex change were compared for brain mRNA expression of AVT, AVT receptors, and GnRH to determine the order of hormonal change during the process of sexual inversion in this species. This study provides a better understanding of the relationship between sex-specific behavior and reproductive development via AVT and GnRH systems that are conserved in all vertebrates. / text

The effects of vitamin E (VE) and Arginine (ARG) on humoral and cellular immunity in chickens were investigated in two experiments. The humoral immunity was measured by antibody responses to sheep red blood cells (SRBC) and maternal antibody titers to the infectious bursal disease virus (IBDV), while the cellular immunity was studied using the cutaneous basophil hypersensitivity test to phytogemagglutinin (PHA) and by counting subpopulations of T-lymphocytes. We used two levels of ARG: normal (NARG, 1.2% in feed) and high ARG (HARG, additional 0.3% in drinking water or 1% in feed in experiments 1 and 2, respectively); and three levels of VE were given: 40, 80, and 400 IU/kg feed in experiment 1, and 40, 80, and 200 in experiment 2. / HARG improved the antibody response to SRBC compared with NARG ( P<0.01 for experiment 1 and P<0.013 for experiment 2) 4 days after injection in both experiments. In experiment 1, the VE80 birds maintained higher antibody titers to SRBC (P<0.001) than the VE40 and VE400 birds 4, 8 and 16 d after inoculation. In experiment 2, the antibody titers to SRBC were higher in the VE80 birds compared with the VE200 birds at days 5, 8, and 12 after inoculation (P<0.001). Maternal antibody titers (log10) to the IBDV were higher in the HARG than in the NARG diet in 17-day-old birds (P<0.001) and higher in the VE80 than in the VE40 birds (P<0.001), yet similar to those of the VE200 birds. No interactions were found between ARG and VE. / Naive birds fed HARG exhibited a higher response than NARG birds (P<0.05) to PHA-P at d 17 and to PHA-M at d 41, but, after a second exposure, high ARG levels did not have an effect. Also, in naive birds, the effects of VE were not significant at d 17, but showed an influence after a second exposure in 41-d-old birds. / The percentage of T-helper (Th) and T-cytotoxic (Tc) cells in the blood of 29-d-old birds were not different between ARG levels (P=0.07 and P=0.06, respectively), but Th cells were higher in the VE80 and VE200 birds than in the VE40 birds, and Tc was higher in the VE80 than in the VE40 birds (P=0.02). The B-cell:T-cell ratio was higher in the HARG than the NARG birds (P=0.01) and in the VE40 compared with the VE80 and VE200 birds (P<0.001). Neither ARG nor VE had an effect on the ratio of Th:Tc cells, nor on the percentage of immature T-lymphocytes. / A combination of high levels of ARG and high levels of VE (80 IU/kg of BW) has an important immunomodulation effect on the cellular and humoral immune responses in broiler chickens, improving both maternal antibody titers against the IBDV and antibody titers against SRBC. A combination of ARG and VE increases the proportions of Th and Tc cells, the B-cell:T-cell ratio, and growth performance. The evidence suggests that ARG and VE play complementary and regulatory role on immune response and may enhance the resistance of broilers to infectious diseases. / Key words. Arginine, vitamin E, humoral immunity, cell-mediated immunity, lymphocyte, ELISA. / L'effet de la vitamine E (VE) et l'arginine (ARG) sur les systèmes hummoraireet cellulaire de l'immunité a était évalué chez la volaille dans deux recherches. Lesystème hummoraire de l'immunité a était évalué en utilisant les paramètres tels que laproduction d'anticorps après une injection des globules rouge provenant des moutons(SRBC) et le niveau d'anticorps maternelle après une infection avec les virus causantla maladie 'infectious bursal disease' (lBDV), tandis que les effets sur le systemcellulaire de l'immunité avaient aussi été évalués en utilisant les paramètres comme'cutaneous basophil hypersensitivity test to phytogemagglutinin (PHA)' et endéterminant la concentration des lymphocytes T. Deux concentrations de ARG avaientété utilisées: normale (NARG, 1.2 % de la diète) et une concentration élevée (HARG,additionel 0.3 % dans l'eau ou 1 % dans les diètes); et 3 concentrations de VE: 40, 80et 400 lU/kg dans les diètes dans la première recherche et 40, 80, et 200 lU/kg dans ladeuxième recherche.

Background: Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease with symptoms of polydipsia, polyuria and dehydration caused by arginine vasopressin deficiency. Disease onset is within infancy or adolescence. A variety of disease-causing mutations of the arginine vasopressin neurophysin II gene (AVP) on chromosome 20p13 have been described. Methods: Two Polish families with adFNDI were screened for mutations. Processing of wild-type (WT) and mutant AVP was monitored using immunocytochemical methods in stably transfected Neuro2A cells. AVP secretion into the cell culture supernatant was investigated with an enzyme immunoassay. Results: In the first family a heterozygous p.G96D mutation was identified. Some patients additionally carried a novel heterozygous mutation p.A159T. The second family presented with a heterozygous mutation p.C98G. Confocal laser microscopy unveiled accumulation of p.G96D and p.C98G prohormones in the cellular bodies, whereas WT and p.A159T prohormones and/or processed products were located in the tips of cellular processes. Reduced levels of AVP in supernatant culture medium of p.G96D and p.C98G transfected cells in comparison to p.A159T and WT cells were found. Conclusions: We conclude that the p.G96D and p.C98G mutations cause adFNDI in the two reported families. The sequence variant p.A159T does not seem to have disease-causing effects. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Spinal Muscular Atrophy (SMA) is a neuronal degenerative disease caused by the mutation or loss of the Survival Motor Neuron (SMN) gene. The cause for the specific motor neuron susceptibility in SMA has not been identified. The high axonal transport/localization demand on motor neurons may be one potentially disrupted function, more specific to these cells. We therefore used a large-scale immunoprecipitation (IP) experiment, to identify potential interactors of SMN involved in neuronal transport and localization of mRNA targets. We identified KH-type splicing regulatory protein (KSRP), a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing, and mRNA decay. KSRP is closely related to chick zipcode-binding protein 2 and rat MARTA1, proteins involved in neuronal transport/localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. We demonstrated that KSRP is arginine methylated, a novel SMN interactor (specifically with the SMN Tudor domain; and not with SMA causing mutants). We also found this protein to be misregulated in the absence of SMN, resulting in increased mRNA stability of KSRP mRNA target, p21cip/waf1. A role for SMN as an axonal chaperone of methylated RBPs could thus be key in SMA pathophysiology.

Two identical experiments were conducted to evaluate the combined effects of arginine (AR), vitamin E (VE) and vitamin C (VC) on cardiopulmonary performance and ascites parameters of broilers reared under cold environmental temperature. One d old male broilers were fed a basal corn-soybean meal diet (CTL, 1.2 % AR and 40 IU VE), the basal diet supplemented with 1% AR and either 200 IU VE (AE group), or 500 mg of VC (AC group), or a combination of VE and VC (AEC group) at the same level per kg of feed. Pulmonary arterial pressure (PAP) and mean arterial pressure (MAP) were recorded in clinically healthy anesthetized birds (28-42 d old) before and after an epinephrine challenge (EPI, 0.5 mg/kg BW i.v.), Amino-guanidine Hemisulphate (AG, 100 mg/kg BW i.v.) and N-Nitro L-Arginine Methyl Ester (L-NAME 50 mg/kg BW, i.v.) at 20 min intervals. The PAP increased 30 s after EPI in all birds, but the peak PAP was lower in the AEC group than in all the other birds. After 120 s of challenge the PAP was lower in AEC birds compared to the other birds. The PAP returned to pre-EPI levels within 300 s in all groups. The PAP was increased ( P< 0.05) within 60 s after the AG and L-NAME challenge in all groups, but no differences were found among groups. Plasma nitric oxide (NO) was higher in the AEC birds than in all the other groups before and after challenge. Our results showed that birds fed AEC maintained a lower PAP than the CTL birds after EPI elicited an increase in cardiac output and this can be explained by a higher production of NO. A combination of AR, VE, and VC may have complementary effects against oxidative stress, protecting the endothelium and preserving NO function.

Septic shock is a major cause of deaths resulting from uncontrolled inflammation and circulatory failure. Recent studies suggest that the vascular isoform of ATP-sensitive K+ (KATP) channels is an important contributor to septic susceptibility. To understand the molecular mechanisms for channel regulation during sepsis, we performed studies in isolated endothelium-denuded mesenteric rings. Lipopolysaccharides (LPS) induced vascular relaxation and hyporeactivity to phenylephrine. The LPS-treated aortic smooth muscle cells displayed hyperpolarization and augmentation of KATP channel activity. Both were due to an up-regulation of Kir6.1 and SUR2B surface expression. The up-regulation relied on transcriptional and translational mechanisms, in which nuclear factor-¦ÊB (NF-¦ÊB) and Protein kinase A (PKA) played a critical role. Oxidative stress occurs during sepsis and may act as another regulatory mechanism affecting KATP channel activity and vascular contractility. We found that micromolar concentrations of H2O2 impaired the pinacidil-induced vasodilation. The effect attributed to the suppression of KATP channel activity, which can be fully produced by reactivity oxidants. Unlike the Kir6.1/SUR2B channel, the Kir6.2/SUR2B channel was insensitive to 1mM H2O2, indicating that the modulation sites are located in Kir6.1. Site-directed mutational analysis showed that three cysteine residues located in N-terminus and the core region of Kir6.1 were likely to mediate the redox-dependent channel modulation. Arginine vasopressin (AVP) is a vasoconstrictor that is successfully applied to manage sepsis. However, the downstream target of AVP is uncertain. Our studies show that AVP-induced vasoconstriction depended on V1a receptor, Protein kinase C (PKC) and KATP channel. Additionally, AVP decreased Kir6.1/SUR2B channel activity through V1a receptor. The inhibitory effect was caused by a suppression of the channel open state probability. The channel inhibition was mediated by phosphorylation of the channel protein by PKC. The widespread involvement of the vascular KATP channel in vascular responses to endotoxemia strongly suggests that the temporospatial control of channel activity may constitute an important intervention to vascular tone, blood pressure and organ-tissue perfusion in septic shock. Such a control appears feasible by targeting several modulatory mechanisms of intracellular signaling, Kir6.1/SUR2B expression, redox state and channel protein phosphorylation as demonstrated in this dissertation.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [119]-139).

Thesis (Ph.D. in Microbiology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 201-223).

O objetivo central dos dois estudos que compõem essa tese foi estudar a manipulação de aminoácidos em linhagens hiperprolíficas, nas quais o desafio do crescimento e produção de leite é ainda maior. No primeiro trabalho, foram utilizadas leitoas Landrace x Large White gestantes distribuídas em quatro tratamentos: Argiblend – oferta de 1% de arginina do D25-D80 e de 20g de blend do D81-D112 de gestação; Arginina – oferta de 1% de arginina do D25 ao D80; Blend – oferta de 20g de blend do D81 ao D112, e Controle - dieta controle durante toda a gestação. Não houve efeito (P>0,05) dos tratamentos sobre o peso e ganho de peso das matrizes, bem como para número de nascidos totais, nascidos vivos, natimortos, mumificados, peso ao nascer, coeficiente de variação do peso ao nascer, percentual de leitões ≤850g e ≤1000g, peso de placenta, eficiência placentária e expressão do fator de crescimento do endotélio vascular. Houve efeito da interação do tratamento e classe de prolificidade (P<0,05) sobre os percentuais de leitões leves. Na classe de baixa prolificidade (até 14 leitões nascidos), menores percentuais de leitões com peso ≤850g foram observados nos tratamentos Arginina e Blend em comparação ao Controle (P<0,05). Ainda dentro das fêmeas de baixa prolificidade, o tratamento Blend teve maior peso de leitegada e peso médio de nascidos, além de menor percentual de leitões com peso ≤1000g do que as fêmeas Controle (P<0,05). O uso de suplementação de arginina 1% do D25-D112, blend de aminoácidos do D81-D112 ou a combinação de ambos, não afeta o peso médio ao nascer e nem o coeficiente de variação do peso, em leitegadas de matrizes hiperprolíficas. Porém, arginina e blend diminuem o percentual de leitões leves e blend aumenta o peso médio ao nascer nas leitegadas com até 14 leitões. O objetivo do segundo estudo foi investigar o efeito da suplementação de arginina na dieta de lactação sobre a composição do leite, desempenho e sobrevivência de leitões lactentes. Sessenta e quatro matrizes lactantes de linhagem Landrace x Large White de ciclo 1 a 7 foram distribuídas em dois tratamentos: 1) suplementação diária com 1% de Arginina via top dressing durante toda a lactação e 2) Controle. Não houve efeito do tratamento sobre o peso médio individual, peso da leitegada e ganho de peso diário dos leitões, tanto no D10 como no D20 da lactação (P>0,05). A interação entre o dia da pesagem e o tratamento não afetou nenhuma dessas variáveis (P>0,05). No geral, as taxas de sobrevivência dos leitões foram 90,3% e 88,3% no D10 e D21, respectivamente, sem diferença entre os tratamentos (P>0,05). Não houve efeito do dia da lactação (D10 ou D20), do tratamento ou da interação entre eles, sobre a proteína bruta e conteúdo de aminoácidos do leite (P>0,05). A suplementação de 1% de arginina utilizando top dressing na dieta de lactação de fêmeas suínas, de ciclo 1 a 7, não influencia o desempenho e sobrevivência dos leitões na maternidade e não altera a relação lisina:arginina bem como o teor de proteina e de aminoácidos no leite. / The central objective of both studies was to test the effect of amino acid supplementation in prolific sows, in which fetal growth and milk production is a big challenge.The first study was conducted to evaluate the effect of arginine and/or amino acids blend (lysine, methionine, threonine and tryptophan) supplementation in gestation diet of prolific primiparous sows on the placental efficiency and piglet’s birth weight. Pregnant gilts were distributed into four treatments: Argiblend – supplemented with 1% of arginine from D25 to D80 and 20g of blend from D81 to D112 of gestation; Arginine - supplemented with 1% of arginine from D25 to D80; Blend - 20g of blend from D81 to D112; and Control – basal diet from D25 to D112. There was no treatment effect (P>0.05) on the gestation body weight gain of sows, total number of piglets born, born alive piglets, stillborn piglets, mummified fetuses, average birth weight, coefficient of whithin-litter weight variation, proportion of low birth weight piglets (≤850g or ≤1000g), placental efficiency and vascular endothelial growth factor expression. The effect of amino acid supplementation was also evaluated taking into account the prolificity of females, which were separated in two categories: high (>14 piglets born) and low (≤14 piglets born) prolificity. In low prolificity females, the proportion of piglets with ≤850g was lower in arginine and blend treatments compared with the Control group. In addition, females receiving blend had total litter birth weight and average birth weight greater and the proportion of piglets with ≤1000g lower than in Control group (P<0.05). In the present study, supplementing gestation diets with arginine and/or amino acid blend does not influence the average birth weight and birth weight variation in primiparous prolific sows. However, arginine and blend decrease the proportion of light piglets at birth and increase the average birth weight when the litter size is ≤ 14 piglets. The objective of the second study was to investigate the effect of arginine supplementation in the lactation diet on milk composition and the performance and survival of suckling piglets. Sixty four lactating Landrace x Large White sows, parity 1 to 7, were assigned in two treatments: Arginine – control diet supplemented daily with 1% arginine by top dressing during all lactation, and Control – control cornsoybean based diet. The sows remained with 12.8 piglets on average after cross fostering. The litters were weighed on D1, D10 and D21 of lactation and piglet mortality was recorded. Samples of milk (60 ml) were collected at D10 and D20 of lactation, from all functional teats. There was no effect of arginine supplementation on the average individual weight, litter weight and average daily gain of piglets at both D10 and D20 of lactation (P>0.05). The interaction between the day of weighing and treatment did not affect any of these parameters (P>0.05). Overall, survival rates were 90.3% and 88.3% until D10 and D21, respectively, with no difference between treatmens (P>0.05). There was no effect of the lactation day (D10 or D20), treatment or the interaction between them on the crude protein and amino acid content in milk (P>0.05). Supplementation with 1% of arginine by top dressing, in lactation diet of parity 1 to 7 sows, does not affect the performance and survival of piglets, as well as does not alter the amino acid content or lysine:arginine ratio of milk.

O objetivo deste trabalho foi verificar o possível efeito protetor da L-glutamina e da L-arginina sobre a próstata ventral de ratos quando administradas por gavagem. Procurou-se simular as condições clinicas de pacientes submetidos à radioterapia pélvica tendo como órgão alvo outro órgão pélvico que não a próstata. Foram analisados os efeitos desta irradiação sobre a próstata considerando este órgão como normal. Foram utilizados ratos Wistar divididos em quatro grupos: Controle, animais não submetidos à irradiação (n= 10); Irradiado, submetidos à irradiação abdominal e sem suplementação adicional de aminoácido por 21 dias (n= 10); Irradiado + Lglutamina, submetidos à irradiação abdominal e com suplementação adicional de L- glutamina por 21 dias (n= 10); e Irradiado + L-arginina, submetidos à irradiação abdominal e com suplementação adicional de L- arginina por 21 dias (n= 9). Os grupos foram mantidos em condições padrão de laboratório durante todas as etapas do experimento. Os animais submetidos à irradiação abdominal receberam uma dose única de 1000 cGy no dia 8 da experimentação. A Lglutamina e a L-arginina foram dissolvidas em água destilada e administrada por gavagem através da agulha IC-810. As próstatas foram removidas e processadas para inclusão em parafina. Foram estudados os seguintes parâmetros: estrutura acinar (área dos ácinos e altura do epitélio) e colágeno analisados por métodos morfométricos e peso corporal. O ganho de peso nos grupos suplementados foi significativamente maior se comparado ao grupo irradiado. Houve redução da altura do epitélio no grupo irradiado quando comparado ao controle. A altura do epitélio no grupo suplementado com L-arginina foi significativamente maior do que nos grupos irradiado e suplementado com L-glutamina. Houve diminuição, de aproximadamente 18%, da área dos ácinos no grupo suplementado com L-glutamina. Já no grupo suplementado com Larginina o valor foi similar ao do controle. O efeito da L-glutamina sobre o parênquima prostático foi o de manter proporcionalmente o colágeno, preservando a integridade da matriz extracelular. No grupo suplementado com L-arginina, apesar da discreta redução na distribuição proporcional de colágeno este também manteve índices semelhantes ao do controle. A radiação abdominal promoveu algumas modificações estruturais na próstata ventral de ratos. Essas modificações podem ser parcialmente prevenidas pela suplementação oral com L-glutamina e de L-arginina. / The aim of this study was to investigate the possible protective effect of L-glutamine and Larginine on the rat ventral prostate when administered by gavage. We tried to simulate the clinical conditions of patients undergoing pelvic radiotherapy as with other pelvic organ target organ than the prostate. We analyzed the effects of irradiation on prostate considering this organ as normal. Wistar rats were divided into four groups: Control, animals not exposed to irradiation (n= 10); Irradiated, submitted to abdominal irradiation and without additional amino acid supplementation (n= 10); Irradiated + L-glutamine, submitted abdominal irradiation and additional supplementation with L-glutamine for 21 days (n= 10), and Irradiated + L-arginine, underwent abdominal irradiation and additional supplementation with L-arginine for 21 days (n=9). The groups were kept under standard laboratory conditions during all stages of the experiment. The animals underwent abdominal irradiation received a single dose of 1000 cGy in eight days of trial. L-glutamine and L-arginine were dissolved in distilled water and administered by gavage needle through the IC-810 . The prostates were removed and processed for paraffin embedding. We studied the following parameters: acinar structure (area of acini and epithelial height) and collagen analyzed by morphometric methods and body weight. Weight gain in the supplemented groups was significantly higher compared to the irradiated group. There was a reduction in epithelial height in the irradiated group compared to control. The height of the epithelium in the group supplemented with L-arginine was significantly higher than in irradiated and supplemented with L-glutamine. There was a decrease of approximately 18% of the area of the lobes in the group supplemented with L-glutamine. In the group supplemented with Larginine was similar to the value of control. The effect of L-glutamine on the prostatic parenchyma was to keep the collagen in proportion, preserving the integrity of the extracellular matrix. In the group supplemented with L-arginine, despite a slight reduction in the proportional distribution of this collagen also maintained similar levels to the control. The abdominal radiation caused some structural changes in rat ventral prostate. These modifications can be partially prevented by oral supplementation with L-glutamine and L-arginine.