Historically, sperm has been considered merely as a carrier of genetic material at fertilisation. However, it is known that sperm supports embryonic development better than other cell types, suggesting that it might also have additional important, non-genetic contributions to embryonic development. The work described in this dissertation focuses on identifying the molecular determinants of developmental programming of sperm. First, the development of embryos derived from sperm and spermatids, immature precursors of sperm was compared. Sperm-derived embryos developed significantly better than spermatid-derived embryos. Further research aiming to identify the reasons for the developmental advantage of sperm led to the identification of proteins that are present specifically in sperm and not in spermatids. Moreover, egg factors which are preferentially incorporated into the sperm, but not into the spermatid chromatin were identified with the use of egg extracts, suggesting that the chromatin of sperm could be programmed to interact with the components of the egg. Subsequently, the reasons for developmental failure of spermatid-derived embryos were investigated. By comparing the sperm with spermatids it was shown that the programming of sperm to support efficient development is linked to its special ability to regulate expression of developmentally-important embryonic genes, and not to its ability to support DNA replication or rRNA production. Further characterisation of the sperm and spermatid chromatin with the use of genome-wide sequencing allowed me to link the correct regulation of gene expression in the embryo with a certain combination of epigenetic marks in the sperm, but not in the spermatid chromatin. Finally, it is shown that enzymatic removal of epigenetic modifications at fertilisation leads to misregulation of gene expression. This therefore suggests that epigenetic information contained in parental genomes at fertilisation is required for a proper regulation of embryonic transcription. My results support the hypothesis that the sperm is not only a carrier of genetic material, but also provides the embryo with epigenetic information for regulation of transcription after fertilisation. I believe that these findings advance our current understanding of the nature and mechanisms of sperm programming for embryonic development, and are important contributions to the emerging field of transgenerational inheritance of epigenetic traits in general.

A la suite d’un criblage in silico nous avons identifié un nouveau gène codant pour une protéine à domaine BTB-POZ, XBTBD6.<p>Nous avons déterminé que la protéine XBTBD6 est une protéine cytoplasmique. Dans les cellules Hela, CHO, U2OS et COS7 la protéine XBTBD6 est localisée dans des corpuscules cytoplasmiques, localisation similaire à celle des protéines XBTBD3, HBTBD1 et HBTBD2. Nous avons observé que la partie N-terminale de la protéine, contenant le domaine BTB-POZ, est localisée dans la cellule comme la protéine entière ;par contre la partie C-terminale est exclusivement nucléaire. De plus, nous avons observé que XBTBD6 est localisée de façon diffuse dans le cytoplasme des cellules Neuro2A, 9L et 518A2e. Nous avons montré que la protéine XBTBD6 homodimérise et hétérodimérise avec XBTBD3 et XBTBD2 et qu’elle interagit avec l’ubiquitine ligase E3 XCullin 3. L’ensemble de ces interactions nécessite la présence du domaine BTB-POZ. Ces données montrent que les protéines BTBD6, BTBD3, BTBD1 et BTBD2 possèdent des propriétés communes indiquant qu’elles appartiennent à un sous groupe de la famille des protéines à domaine BTB-POZ.<p>Le profil d’expression a été analysé par la technique de protection à la RNAse et par hybridation in situ. Les résultats montrent que ce gène est fortement exprimé dans le système nerveux adulte et embryonnaire. Des expériences de surexpression par micro-injection d’ARNm ont permis de placer le gène XBTBD6 dans la cascade d’activation des gènes proneuraux en aval de XNgnr-1, XNeuroD, Xath3 et Xebf3. Ces résultats montrent que XBTBD6 est un marqueur neuronal chez le xénope. <p>Au cours de l’étude de la fonction du gène XBTBD6, nous avons montré que la surexpression et la perte de fonction de ce gène dans l’embryon de xénope n’induit pas de variation du nombre de neurones dans la plaque neurale. Par contre nous avons observé que la surexpression du gène XBTBD6 dans des cellules Neuro2A en différentiation régule négativement la croissance des neurites.<p>Nous avons élaboré un modèle de fonctionnement biochimique hypothétique où la protéine XBTBD6 fonctionnerait comme protéine adaptatrice dans un complexe d’ubiquitination permettant l’ubiquitination d’une protéine cible. Nous avons recherché les partenaires potentiels de XBTBD6 en utilisant la technique du double hybride en levure mais sans y parvenir.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Imaging Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) has been developed to perform 2D imaging and depth profiling of biological systems with micron or submicron scale lateral resolution, which can be attributed to the advent of polyatomic ion beam particularly C60+ and new concept of ToF-SIMS instrument, the J105 3D Chemical Imager (J105). These recent advances in ToF-SIMS have opened a new dimension for biological analysis. In this study, 2D and 3D imaging have been performed on two biological systems, Xenopus laevis (X. laevis) zygote/embryo and murine embryonic fibroblasts NIH 3T3 BXB-ER cells to explore the capability of ToF-SIMS to handle the biological samples with extreme topography and high resolution depth profiling of microdomains, which still represent major challenges for the ToF-SIMS. The study on X. laevis embryo explored the capability of ToF-SIMS to handle spherical samples (approx. 1-1.2 mm in diameter), identify lipid species in mixtures of lipid extraction from the zygotes and image of an intact embryo in 2D/3D during dynamic biological events, e.g., fertilisation and early embryo development. For the first time the J105 and conventional BioToF-SIMS instrument were employed for the study of developmental biology. The major classes of lipid were identified through multiple lipid assay in a single analytical run using ToF-SIMS. Topography effects of the embryo were assessed through imaging a single intact zygote/embryo that revealed secondary ions loss at the edge of the single cell. However, the topography effects on the mass resolution could be minimised using the J105. Moreover, in situ lipid profiling of the zygote revealed different lipid compositions and intensities on the membrane of the animal and vegetal hemispheres. Furthermore, high resolution imaging and depth profiling that performed on a single intact cell in a time course study visualised the egg-sperm fusion sites on the membrane of the zygote 10 min post-insemination and lipids arrangement on the membrane of the embryo through the early development stages. Subcellular signalling upon the fertilisation was also spatially located on the serial cryosections of a single zygote. With the NIH 3T3 BXB-ER cells, the study firstly adopted a finely focused C60+ beam to track morphological changes and rearrangement of subcellular organelle mitochondria (0.5-2 µm) in response to the activation of Raf/ERK (extracellular signal regulated kinase) pathway using the J105. The SIMS images of the unlabelled cells showed the shifting of membrane distribution and nuclei shrinking following Raf/ERK activation. The mitochondria fluorescence probe within the cells were located 3-dimensionally using confocal microscopy and ToF-SIMS, which revealed the distribution pattern of condensing in the two sides of the nuclei following the Raf/ERK activation. Coupled with scanning electron microscopy (SEM), the three imaging modes showed good agreement in cellular morphological changes and subcellular mitochondrial rearrangement without or following Raf/ERK activation, demonstrating an integrated approaching to study the biological processes at subcellular dimension.

Le noyau paraventriculaire (PVN) de l'hypothalamus régule une série de phénomènes physiologiques incluant l'équilibre énergétique et la pression artérielle. Nous avons identifié une cascade de facteurs de transcription qui contrôle le développement du PVN. SIM1 et OTP agissent en parallèle pour contrôler la différenciation d'au moins cinq types de neurones identifiables par la production d'OT, AVP, CRH, SS et TRH. Ces Facteurs de transcriptions contrôlent le développement des lignées CRH, AVP et OT en maintenant l'expression de Brn2 qui à son tour est nécessaire pour la différenciation terminale de ces neurones. L'analyse du transcriptome du PVN nous a permis d'identifier plusieurs gènes qui ont le potentiel de contrôler le développement du PVN. Nous voulons développer un paradigme de perte de fonction qui permettrait l'étude de ces gènes candidats sur une grande échelle. Le but de ce projet est de caractériser le PVN en développement de l'amphibien en vue de l'utilisation de ce modèle pour des études fonctionnelles. Nous avons cloné des fragments de cDNA de Sim1, OTP, Brn2, Sim2, CRH, Ot, AVP et TRH à partir de l'ARN total de Xenopus Laevis. Nous avons adapté notre technique d'hybridation in situ pour caractériser l'expression de ces gènes chez l'amphibien aux stades 33-39, 44, 51, 54, 60, et chez l'adulte. Résultats. Les Facteurs de transcription Sim1, OTP, et Brn2 commencent à être exprimés dans le PVN prospectif au stade 33. L'expression des marqueurs de différenciation terminale devient détectable entre les stades 37 et 39. De façon intéressante, le PVN occupe initialement un domaine de forme globulaire puis à partir du stade 44 s'allonge le long de l’axe dorso-ventral. Cet allongement se traduit par une organisation en colonnes des cellules du PVN que nous n'avons pas observée chez les rongeurs. Le développement du PVN est conservé chez l'amphibien dans la mesure où la relation entre l'expression des facteurs de transcription et des marqueurs de différenciation terminale est conservée. Il existe par ailleurs des différences entre la topographie des PVN des mammifères et de l'amphibien. L'organisation en colonnes de cellules pourrait correspondre à des mouvements de migration tangentielle. Nous sommes maintenant en mesure de tester la fonction des facteurs de transcription dans le PVN par l'approche d'invalidation par morpholinos. / The paraventricular nucleus PVN of the hypothalamus regulates a series of physiological phenomena including the maintenance of energetic balance and arterial blood pressure. We have previously identified a cascade of transcription factors that control the development of the PVN. Sim1 and OTP act in concert to mediate the terminal differentiation of at least five types of neurons identifiable by their production of OT, AVP, CRH, SS and TRH. These transcription factors control the development of the OT, AVP and CRH producing neurons by maintaining the expression of Brn2, which is in turn required for the terminal differentiation of these cell lines. The transcriptome analysis of the PVN allowed us to identify a handful of genes that are potentially implicated in the development of this brain structure. Our goal is to develop a loss of function paradigm that would allow a high troughput study of these candidate genes. The main goal of this project is to characterize the developing PVN in the amphibian in order to use this model in our functional studies of these genes. We have cloned fragments of cDNA of Sim1, OTP, Brn2, Sim2, CRH, TRH, AVP and OT using Xenopus laevis total RNA. We have also adapted our in situ hybridization technique to characterize the expression of these genes in stage 33-39, 44, 51, 54, 60 and adult amphibian brain. Sim1, OTP and Brn2 are expressed in the prospective PVN as soon as stage 33. The expression of the terminal differentiation markers become detectable between stages 37-39. Interestingly, the PVN is initially restricted to a more globular domain and begins to extend along the dorso-ventral axis at around stage 44. This vertical extension translates into a column organization that we do not observe in rodents. The development of the PVN is well conserved in the amphibian in the sense that the relation between the expression of the different transcription factors and the terminal differentiation markers is conserved. We can also observe some topographical differences between the mammalian and amphibian PVN. The column organization the different PVN cell types might correspond to the tangential migration that is observed in the mouse. We are now well equipped to test the function in the PVN of the known transcripton factors as well as the candidate genes previously identified in our lab using a morpholino-mediated gene knock down.

M.Sc. / The increase in industrialisation, mining and agricultural activity along rivers could have a detrimental effect on aquatic environments unless the dangers of pollutants are not taken notice of. Metal ions and industrial effluent have become a source of pollution in the watercourses of South Africa. Pollutants generally have negative effects on the physiology of aquatic biota in polluted waters. The effects of copper and industrial effluent by the exposure of the clawed toad, Xenopus laevis and Tilapia sparrmanii are presented in this study. An experimental static-renewal system with an exposure time of 96 hours was followed at 25±1°C. After copper and effluent exposure, several physiological changes occurred in the two aquatic organisms. The sublethal effects that occurred include changes in hatching, survival, behaviour, growth impairment and developmental limitations. The results of the present study suggest that lethal endpoints can be used as indicators in detecting and evaluating the effects of aquatic pollution, caused by copper and effluent. Individual variation, however, could hamper the conclusions made but the study of aquatic organisms is of practical importance when conducting experimental studies in a laboratory and does not have the same impact as during field studies. Apart from the exposure to sublethal concentrations of copper and effluent, computational derivations of LC50, NOEC values and 95% confidence limits were made. The obtained concentrations were used as assumptions that pollutants should not exceed for the protection of aquatic life. Statistically different differences were found between the chosen derived variables of control and experimental organisms. The advantages of FETAX solution over borehole water can be attributed to the bioavailability of pollutants, which appears to be much less in those solutions. The predicted NOEC values provide some information regarding the concentrations at which no effects will be observed and the Target Water Quality Ranges (TWGR) for water were used to determine if the diluent was correct.

The amphibian decline phenomenon now involves in excess of a third of the roughly 6000 species of amphibians on the planet. The problems that drive the declines are diverse with no end in sight. The Amphibian Conservation Action Plan (ACAP) aims to stem amphibian decline through four recommended actions by researchers and conservation biologists: (1) Expand scientific understanding of amphibian declines and extinctions; (2) continue to document amphibian diversity and ecology and how they are changing; (3) develop and implement long-term conservation programs; (4) prepare emergency response actions for eminent crises. This Dissertation focused on two of those recommendations: expanding scientific understanding of amphibian declines and extinctions and continuing to document amphibian diversity and ecology and how they are changing. The first chapter is a review of the amphibian decline phenomenon. The second, third, and fourth chapters focus on expanding scientific understanding of amphibian diversity and ecology with the description of a formerly unknown species (chapter 2), and ecological papers on two poorly known species (chapters 3 and 4). Chapter five focuses on the first ACAP recommendation in improving scientific understanding of the causes behind amphibian decline. The chapter is an experimental examination of two related species and their developmental reactions to common heavy metal contaminants. The goal of this Dissertation is to contribute toward the general amphibian knowledge base relative to the recommendations of ACAP.

La nucleoplasmina (NP) és la proteïna majoritària del nucli dels oòcits i ous no fecundats de la granota "Xenopus laevis" i altres amfibis. Es tracta d'una proteïna acídica amb tres trams acídics principals (A1, A2, A3). La NP participa en la remodelació de la cromatina durant la fecundació, mediatitzant la descondensació de la cromatina espermàtica i facilitant l'acoblament de nucleosomes gràcies a la seva funció de xaperona molecular. En el decurs d'aquest treball s'ha continuat l'estudi dels diferents recombinants de la NP de què disposem, posant especial èmfasi en la forma mutant r-NP142, que es caracteritza pel fet que té el tram acídic principal A2 molt exposat. Interessava determinar la influència de diferents condicions en l'estabilitat i el grau de pentamerització de la molècula. En aquesta mateixa línia es va estudiar el paper de les Cys de la NP en la pentamerització. Mitjançant mutagènesi dirigida es van substituir les Cys per Ser i es va observar que concretament la mutació de la Cys 45 afavoria la monomerització de la forma r-NP142, però no afectava l'oligomerització de les formes r-NP i r-NP121, les quals romanien pentamèriques. Aquests resultats suggereixen que no hi hauria ponts disofre en l'estructura pentamèrica de la nucleoplasmina. La caracterització estructural de la nucleoplasmina es va intentar abordar a diferents nivells. A nivell de l'estructura secundària, es van utilitzar tècniques de dicroisme circular. Concretament es va determinar que el mutant r-NP142 C45S tenia una estructura plegada a l'atzar (random coil). D'altra banda, per a ordres estructurals superiors es van utilitzar tècniques de microscòpia electrònica i tècniques cristal·logràfiques. Un altre objectiu d'aquest treball va ser la caracterització de les interaccions de l'NP amb histones del nucli del nucleosoma. Estàvem especialment interessats a veure si la interacció NP-histones era fonamentalment de tipus electrostàtic o bé podria tractar-se d'algun altre tipus d'interacció. Mitjançant ultracentrifugació analítica i gradients de sacarosa es va poder determinar que la NP podia unir els quatre tipus d'histones nucleosòmiques. Aquesta unió tindria una estequiometria corresponent a 1 mol de pentàmer de NP per mol d'octàmer d'histones. Cal destacar que la NP pot unir també histones tripsinitzades (sense les cues bàsiques) mantenint la mateixa estequiometria que per al cas de les histones natives. Així mateix, la forma r-NP121 (sense el tram acídic A2) és capaç d'unir histones natives amb la mateixa estequiometria descrita per als casos anteriors. Aquests experiments indiquen que la interacció NP-histones no seria predominantment electrostàtica. Un altra línia de treball va ser estudiar la presència o no d'una NP-like a l'extracte d'oòcits de l'estrella de mar "Echinaster sepositus". S'ha trobat una proteïna majoritària que presenta les propietats de ser termoestable, acídica i parcialment resistent al sulfat amònic. Aquesta proteïna és reconeguda pels anticossos policlonals antinucleoplasmina obtinguts en ous de gallina. D'altra banda, no és capaç de descondensar nuclis espermàtics que contenen protamina.El darrer objectiu del treball va ser l'estudi estructural de la proteïna ?0. La ?0 és específica del nucli espermàtic de l'equinoderm "Holothuria tubulosa" i presenta propietats intermèdies entre les protamines i les histones. La cristal·lització d'una molècula d'aquestes característiques aportaria informació sobre l'estructura de les proteïnes que participen en la compactació de la cromatina i podria obrir un nou camí per a la cristal·lització de les protamines i dels complexos NP-protamina. / Nucleoplasmin (NP) is the most abundant protein found in the nuclei of oocytes and nonfertilized eggs of "Xenopus laevis" and other amphibians. It is an acidic protein with three main acidic tracts (A1, A2, A3). NP is involved in the remodelation of chromatin during fecundation, facilitating nucleosome assembly as a molecular chaperone.In the present work we have continued the study of different recombinant forms of nucleoplasmin which were obtained in our lab, specially the form r-NP142 which has the main acidic tract A2 very exposed. We were interested in how different conditions could affect the stability and pentamerization of the molecule. In this way we studied the importance of Cys in NP pentamerization. We used site directed mutagenesis in order to change Cys for Ser. We observed that the mutation of Cys 45 favoured the monomerization of r-NP142, but this change didn't affect the oligomerization of the forms r-NP and r-NP121, which remained pentameric. These results suggest the absence of disulfide bridges in the pentameric structure of nucleoplasmin. The structural characterization of nucleoplasmin has been studied at different levels. At the secondary structure level we have used circular dichroism techniques and we have determined that the mutant r-NP142 C45S is in random coil conformation. For higher structural levels we have used electron microscopy and crystallographic techniques.Another objective of this work was the characterization of the interactions between NP and nucleosomal core histones. We were specially interested in knowing if the interaction was fundamentally electrostatic. Using analytical ultracentrifugation and sucrose gradient experiments we could determine that NP can bind the four types of nucleosomal histones. This binding would have an stoichiometry equal to 1 mol of NP pentamer per mol of histone octamer. It is important to note that NP can bind trypsinated histones (without the basic tails) maintaining the same stoichiometry as in the case of native histones. In the same way, the r-NP121 form (without the acidic tract A2) is capable of binding native histones with the same stoichiometry described for the other cases. These result indicates that the interaction NP-histones is not mainly electrostaticaly driven.Another objective of this work was the study of the presence of some NP-like proteins in oocyte extracts of the starfish "Echinaster sepositus". We have found a very abundant protein which is termoestable, acidic and partially resistant to ammonium sulphate. This protein is recognized by policlonal antinucleoplasmin antibodies which were obtained in hen eggs. However, it is not able to decondense spermatic nuclei which contain protamine.The last objective of the present work was the structural study of the protein ?0 which is specific of the nuclei of the echinoderm "Holothuria tubulosa" and has intermediate properties between histones and protamines. The crystallization of a molecule like ?0 could bring information about the structure of proteins that participate in chromatin condensation and could offer a new way for the crystallization of protamines and NP-protamine complexes.

Els oòcits de vertebrats es troben aturats a la profase I de la primera meiosi (PI). Durant el procés anomenat oogènesi, els oòctits sintetitzen i emmagatzemen grans quantitats d'ARN missatgers(ARNm)que els seran necessaris per la compleció de la meiosi.I,per posteriorment, aturar-se de nou a la metafase de la segona divisió meiòtica (MII) per l'activitat del factor citostàtic(CSF).D'aquestes divisions en destaca el fet que transcorren en absència de transcripció, i per tant depenen totalment en l'activació traduccional dels ARNm anteriorment esmentats que han estat acumulats durant l'oogènesi. L'activació traduccional d'aquests missatgers és principalment induïda per l'elongació de les cues d'adenines(cues de poli(A)), aquest procés és mediat per les seqüències de poliadenilació citoplasmàtiques (CPE)presents a la regió 3' no tradudïda (3'UTR)dels ARNm. El moment i la longitud de la poliadenilació dels ARNm que contenen CPEs estan finament regulats, de manera que en combinació amb la degradació de proteïnes, s'estableixen els patrons específics d'expresió de les proteïnes que condueixen la meiosi (Shmitt et al., 2002; de Moor and Richter, 1997; Ballantyne et al., 1997; Mendez et al., 2002; Charlesworth et al., 2002). Fins a la data, no s'havia descrit que la deadenilació (escurçament de la cua de poli(A)) fos necessària per la progressió meiòtica. En aquesta tesi s'ha descrit, a partir d'un cribatge d'abast genòmic, una ruta de retroalimentació negativa requerida per a la sortida de la primera metafase meiòtica. La nova ruta identificada, a més té la particularitat d'actuar a nivell traduccional regulant l'expressió de proteïnes que participen directament en la progressió meiòtica. L'element central d'aquesta nova ruta és la proteïna C3H-4, que a la vegada és regulada per poliadenilació citoplasmàtica. C3H-4 crea la retroalimentació negativa interaccionant amb elements ARE de les regions 3'UTR, promovent la deadenilació del ARNm al qual s'uneix. D'entre les seves dianes hem identificat Emi1 i Emi2, ambdós reguladors de l'activitat de l'APC/C, crítica per la divisió cel·lular.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.