Abstract Previous studies have examined stress-induced heme oxygenase-1 (HO-1) expression primarily in mammalian systems. The present study examines, for the first time in amphibians, the effect of heat shock, sodium arsenite, cadmium chloride, and the proteasomal inhibitor MG132 on HO-1 accumulation in Xenopus laevis A6 kidney epithelial cells. Western blot analysis revealed that exposure of A6 cells to a range of heat shock temperatures (30-35 °C), which induced HSP30 accumulation, did not induce HO-1 accumulation. In contrast, cells treated with sodium arsenite (5-50 μM), cadmium chloride (50-200 μM) or MG132 (5-30 μM) exhibited a dose- and time-dependent accumulation of HO-1. Additionally, immunocytochemical analysis revealed that HO-1 and HSP30 accumulation occurred in a granular pattern primarily in the cytoplasm in cells treated with sodium arsenite, cadmium chloride, or MG132. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 and HSP30 accumulation initially increased to a maximum at 12 h and 24 h recovery, respectively, followed by a 50% reduction at 48 h. This initial increase in the relative levels of stress proteins was likely the result of new synthesis as it was inhibited by cycloheximide. In comparison, cells recovering from MG132 treatment displayed reduced but prolonged accumulation of HO-1 and HSP30. Interestingly, cells treated with low concentrations (10 μM) of sodium arsenite or MG132 but not cadmium chloride in combination with a mild 30 °C heat shock had enhanced accumulation of HO-1 and HSP30 accumulation compared to either of the stressors individually. This study has shown for the first time in amphibians that HO-1 accumulation is induced in response to metals and proteasomal inhibitors, suggesting that it may play a role in mediating the cellular stress response in X. laevis.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

En eskalerande minskning hos grodpopulationer och grodarter har skett världen över sedan 1980. Minskningen har påverkats av flera faktorer, bland annat utsläpp av endokrina ämnen till vattendrag. Endokrina ämnen är naturliga och syntetiska hormoner och kemikalier som stör människors och djurs hormonsystem. Syntetiska hormoner finns i p-piller och andra preventivmedel och hormonerna släpps ut i vattendrag via urinen. Det har visats att grodor påverkas av de syntetiska hormonerna etinylestradiol och levonorgestrel som finns i p-piller. Den här litteraturstudien syftar till att undersöka vilka effekter de två hormonerna har på modellarterna Xenopus laevis och Xenopus trolpicalis och på svenska grodarter såsom Rana temporaria. Effekterna är främst förändrad könskvot, med honor i majoritet, samt minskad fertilitet. Fertiliteten försämras genom missbildade könsorgan, genom att äggen mognar långsammare och honor kan bli sterila med avsaknad av äggledare. Hormonexponering kan leda till försenad metamorfos och det kan bli en högre mortalitet hos juvenila grodor. Även hormonuttryck kopplade till reproduktion och fertilitet kan påverkas av hormonexponering. Dock behövs mer forskning, särskilt i fält då mestadels experimentella studier gjorts hittills i laboratorier. Hormonutsläpp är en bidragande faktor till att sex av Sveriges åtta grodarter är rödlistade och i världen handlar det om tusentals arter. Även den så kallade cocktail effekten, det vill säga de effekter flera olika ämnen skapar tillsammans, bör undersökas närmare.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [136]-163).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [76]-93).

O ritmo diário de atividade é uma característica de todos os organismos vivos, que tem a capacidade de se orientar no tempo e no espaço, e distinguir entre tempo linear e tempo cíclico. O ciclo claro:escuro é um importante indicador circadiano para todos os organismos. O trabalho do relógio circadiano envolve mecanismos de retroalimentação positiva e negativa dos genes CLOCK e BMAL1 (brain and muscle Arnt-like protein 1) que formam um heterodímero, funcionando como fator de transcrição para a expressão dos genes per (period), cry (cryptochrome) e o receptor órfão REV-ERB. Em geral, o ciclo circadiano tem início nas primeiras horas da manhã com a ativação da transcrição de per e cry por CLOCK/BMAL1. A periodicidade do relógio circadiano resulta da combinação entre retroalimentação transcricional positiva e negativa destes genes. Hoje já se sabe que os vertebrados, além do relógio central (NSQ) possuem vários relógios, distribuídos pelo corpo, os chamados relógios periféricos. A resposta ao estímulo luminoso é resultado da interpretação da informação luminosa por diferentes tipos celulares. A molécula fotorreceptora de melanóforos dérmicos embrionários de X. laevis foi denominada melanopsina (Opn4/Opn4). Neste anfíbio, cones e bastonetes, continuam a exibir ritmo circadiano em cultura durante vários dias, e a sua capacidade de se ajustar pelo estímulo luminoso indica a presença do sistema circadiano. Os objetivos deste projeto foram: verificar qual é o padrão de expressão para Opn4, per1, per2, bmal1 e clock em melanóforos de X. laevis submetidos a diferentes fotofases; verificar se a expressão para Opn4, per1, per2 ,bmal1 e clock nos melanóforos de X. laevis é modulada pela melatonina. Opn4, per1, per2 ,bmal1 e clock Dados obtidos no presente estudo demonstram que nesta linhagem celular estes genes apresentam um padrão de expressão aparentemente rítmico, quando estas células são expostas a um ciclo claro:escuro (14C:10E), que difere do padrão obtido quando mantidas em regime de escuro constante. Em geral, estas células mantidas em escuro constante durante 5 dias tendem a apresentar aumento de expressão de RNAm para estes genes e, quando mantidas em escuro constante também durante 5 dias, mas com adição de melatonina por 1h, 24 h antes de sua extração, estes níveis de RNAm tendem a diminuir. Porém, quando comparamos as três situações, podemos observar que a adição da melatonina restaura, em geral, o padrão de expressão dos genes analisados em 14C:10E. O conjunto de resultados, que obtivemos em melanóforos dérmicos de Xenopus laevis, sugere que esta linhagem celular possue características de relógio periférico. / The daily rhythm of activity is a characteristic of all living organisms, which have the ability of to behave accordingly time and space, and distinguish between linear and cyclic time. The dark:light cycle is an important time cue for all organisms. The work of circadian clock involves mechanisms of positive and negative feedback of CLOCK and BMAL1 which as a heterodimer act as a transcription factor for the expression of per (period), cry (cryptochrome) and the orphan receptor REV-ERB. A typical circadian cycle begins in the first hours of daytime, which the activation of the transcription of per and cry by CLOCK/BMAL1. It is well known that the vertebrates, besides the central clock (SCN), have several other clocks distributed by the body, the so called peripheric clock. The responses to light are the result of the interpretation of light signal by several cell types The photoreceptor molecule in the dermal melanophores of X. laevis was denominated melanopsin (Opn4/Opn4). In this amphibian, rods and cones maintain circadian rhythm during several days in culture, and their ability to synchronize by light suggest the presence of a circadian system. The objectives of this project were: verify the expression pattern for Opn4, per1, per2 ,bmal1 e clock in dermal melanophores of X. laevis, under different photo phases; and verify whether the expression for Opn4, per1, per2, bmal1 and clock were modulated by melatonin. Our data show that these genes have a rhythmic pattern expression, when these cells are under a 14L:10D, which is different from the pattern exhibited in constant dark. In general, these cells in constant dark have a higher mRNA expression, and in the same condition, but with melatonin applied for 1h, 24h before the data collect, these mRNA levels are lower. However, when we compared these three different experimental conditions, we observed that melatonin resets, in overall, the expression pattern of 14L:10D. These data, taken together, suggest that Xenous laevis dermal melanophores have characteristics of a peripheric clock.

No description available.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Heparan sulphate (HS) is critical for embryonic development with involvement in a myriad of biological processes, centrally mediating morphogenic movements and facilitating the specification and differentiation of tissues. Complicated by its structural micro-heterogeneity along with expression on numerous different proteoglycan cores, the plethora of roles for HS in biology and their underlying mechanisms have not yet been fully defined. The discovery and characterisation of new reagents and methods for modification of HS expression and/or structure will aid efforts in elucidating the structure and activity of this glycosaminoglycan. Until now, azido sugars have been utilised as labelling reagents for various types of glycosylation, including N-glycans, O-linked mucin-type glycosylation and O-GlcNAcetylation of proteins. Incorporation of the unnatural azido sugar into the glycan of interest inserts a chemically reactive abiotic azide for subsequent detection via Staudinger ligation or click chemistries. However, to our knowledge, application of these azido sugars has not been explored for glycosaminoglycans. A metabolic labelling approach using Ac4GalNAz yields UDP-GalNAz and UDP-GlcNAz (Boyce et al., 2011), ready to target CS/DS and HS, respectively. We hypothesised that HS synthesis might be altered in the presence of UDP-GlcNAz due to the location of the azide on the acetyl group and the potential for interference with endogenous N-deacetylase-N-sulphotransferase biosynthetic enzyme activity. In mammalian cell culture (Chinese hamster ovary cells), treatment with Ac4GalNAz led to a decrease in total HS abundance accompanied by significant increases in 6-O-sulphation within the chains. Incorporation of a radiolabelled metabolic precursor revealed that average HS chain length was decreased in azido sugar-treated CHO cells. The modifications to HS were dose-dependent and HS inhibition was transient. Following removal of Ac4GalNAz from cell culture, HS expression returned to baseline levels within 24 hours. Previous work from the Bertozzi group has demonstrated the utility of Ac4GalNAz for visualising GalNAc- and O-GlcNAc-modified proteins in vivo. Using Xenopus, we were able to show that treatment of fertilised eggs with Ac4GalNAz decreased the abundance of HS in a similar way to that seen in vitro, with an associated impact on embryonic development. Embryonic axial elongation was impaired, with defective myotomal development and aberrant axonal patterning along the trunk and tail. Posterior somite cell nuclei were disorganised, with loss of distinct chevron patterning and skeletal muscle development was impaired with muscle fibres spanning some of the somite boundaries. Removal of the inhibitor partially rescued tail extension defects, as well as muscle development, but not axonal patterning. Therefore, these experiments illustrate a novel application for Ac4GalNAz as a soluble and reversible inhibitor of HS synthesis for in vitro and in vivo studies. The observed potential for control of inhibition via time- and dose-dependent effects enables targeted and selective inhibition of HS and potentially provides a powerful new inhibitor for the study of HS-mediated events.