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Lfc is a Rho specific guanine nucleotide exchange factor (GEF) that is bound and inhibited by the microtubule (MT) cytoskeleton. In epithelial cells, Lfc promotes actomyosin contractility in response to MT depolymerization; however, its role in leukocytes has not been assessed. Through genetic ablation, we generated an Lfc knockout mouse (Lfc-/-) and tested biochemical and cell biological responses to MT depolymerization in bone marrow derived cells. Lfc was necessary for characteristic actomyosin based contractile behaviours of neutrophils and macrophages, in response to MT depolymerization. Gout is a painful arthritic inflammatory disease, caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a MT-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, blocks neutrophil infiltration to sites of MSU crystal-induced inflammation. We found that Lfc was necessary for the ability of colchicine to inhibit MSU-induced neutrophil infiltration in two in vivo models of gout-like inflammation. Efficient recruitment of leukocytes from the vasculature is a critical step in the immune response to infection. Leukocyte extravasation, which includes rolling, crawling, and diapedesis across the endothelial barrier, is enhanced by fluid shear stress. Through comparison of Lfc+/+ and Lfc-/- mice, we found that Lfc was necessary for in vivo leukocyte crawling and emigration out of the vasculature. Lfc-/- mice also showed defective neutrophil infiltration in response to acute inflammatory insults, and increased mortality in response to polymicrobial infection. In vitro, we found that Lfc was necessary for neutrophil responses to shear stress.

Lfc is a Rho specific guanine nucleotide exchange factor (GEF) that is bound and inhibited by the microtubule (MT) cytoskeleton. In epithelial cells, Lfc promotes actomyosin contractility in response to MT depolymerization; however, its role in leukocytes has not been assessed. Through genetic ablation, we generated an Lfc knockout mouse (Lfc-/-) and tested biochemical and cell biological responses to MT depolymerization in bone marrow derived cells. Lfc was necessary for characteristic actomyosin based contractile behaviours of neutrophils and macrophages, in response to MT depolymerization. Gout is a painful arthritic inflammatory disease, caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a MT-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, blocks neutrophil infiltration to sites of MSU crystal-induced inflammation. We found that Lfc was necessary for the ability of colchicine to inhibit MSU-induced neutrophil infiltration in two in vivo models of gout-like inflammation. Efficient recruitment of leukocytes from the vasculature is a critical step in the immune response to infection. Leukocyte extravasation, which includes rolling, crawling, and diapedesis across the endothelial barrier, is enhanced by fluid shear stress. Through comparison of Lfc+/+ and Lfc-/- mice, we found that Lfc was necessary for in vivo leukocyte crawling and emigration out of the vasculature. Lfc-/- mice also showed defective neutrophil infiltration in response to acute inflammatory insults, and increased mortality in response to polymicrobial infection. In vitro, we found that Lfc was necessary for neutrophil responses to shear stress.

The feasibility and characterization of a novel diaphragmless shock tube was examined at the National Aerothermochemistry Laboratory at Texas A&M University. The goal was to design a facility that reliably produces shock waves through air in a repeatable manner sufficient for statistical analysis. The device is modular, automated, and compact. The proposed diaphragmless shock tube uses a shock wave generating mechanism that consists of a rotating door and locking cam-shaft system. The facility produced the desired driver gas pressures repeatedly to within 0.31% at low-duty-cycle of 6 seconds. The driven gas pressure profiles within the test-section suggest that shock waves may be forming within test section for a driver gas pressure of 200 psig and above, which corresponds to shock wave Mach numbers of 1.7 to 2.0. The measured wave speeds were within 3.1% of that predicted by ideal shock tube theory; however, the induced driven gas pressures within the constant pressure region were approximately half that expedited from ideal shock tube theory.

El shock es una situación clínica que se ha venido observando durante cientos de años y cuya presencia ha ido asociada con procesos que culminaban casi siempre con la muerte. El estudio de estos procesos ha permitido llegar a la conclusión de que el shock tiene un componente multifactorial y multietiológico. La cascada de eventos que tienen lugar en un estado de shock parece demasiado compleja como para que la actuación terapéutica sobre uno solo de estos pasos sea suficiente como para bloquear el shock en su conjunto. Sin embargo, desde hace años se vienen estudiando diferentes aproximaciones terapéuticas entre las que se encuentra el antagonismo del factor activador de ls plaquetas (PAF). Los diferentes trabajos desarrollados en esta tesis pretenden ser una aportación al estudio del papel del PAF y de sus antagonistas en el shock. Para ello se han recogido una serie de modelos experimentales, tanto in vitro como in vivo , con el antagonista del PAF UR-12460. Los modelos estudiados son: shock anafiláctico, shock por isquemia-reperfusión esplácnica y coronaria, shock endotóxico y shock hemorrágico. El UR-12460 presenta actividad en modelos de shock anafiláctico activo, tanto en cobayo como en ratón. Además, para una serie de antagonistas del PAF estudiados, se demuestra una buena correlación entre la inhibición de la mortalidad inducida por PAF y la inhibición de la mortalidad inducida por shock anafiláctico activo en ratón, lo cual indica que el PAF está implicado en estos procesos. La ineficacia del UR-12460 en procesos de shock pasivo sugiere una menor importancia relativa del PAF con respecto a otros mediadores como la histamina. Por otra parte, en modelos de shock por isquemia-reperfusión esplácnica en rata, el UR-12460 se ha mostrado parcialmente efectivo, inhibiendo la mortalidad y la trombocitopenia producidas por el shock. Otro modelo de isquemia-reperfusión, pero a nivel miocárdico en este caso, ha sido extensamente estudiado en la rata. La oclusión durante 6 minutos de la arteria coronaria izquierda en la rata anestesiada, y posterior reperfusión durante 10 minutos, produce una serie de alteraciones del ritmo cardíaco que son inhibidas por el UR-12460. Dicho compuesto disminuye la incidencia de taquicardias ventriculares durante la oclusión, y disminuye la aparición de fibrilación ventricular en la reperfusión. Esto apoya el papel del PAF en la arritmogénesis por isquemia-reperfusión. Las pruebas de isquemia-reperfusión en corazón aislado de rata, por el método de Langendorff, combinadas con la incubación posterior del tejido cardíaco con cloruro de trifeniltetrazolio, permiten aunar en un mismo experimento y bajo las mismas condiciones, la valoración macroscópica del área de infarto y la alteración funcional del corazón. En este modelo los corazones se perfunden con sangre diluida en líquido de Krebs y se comparan con corazones que reciben sólo el diluyente. Las marcadas diferencias entre los corazones isquémicos que han recibido sangre diluida y los que no, sugieren que las células sanguíneas son pieza clave en la génesis de los infartos y en el deterioro del funcionalismo cardíaco. Por otra parte, la ausencia de una relación directa entre el tiempo de isquemia-reperfusión y la gravedad del infarto, en ausencia de sangre, sugiere que la situación de anoxia no es un factor determinante en la génesis del infarto. Sí existe relación entre el tiempo de isquemia-reperfusión y las alteraciones de la función mecánica. La presencia de células sanguíneas podría representar un sistema de amplificación del daño causado por la anoxia. Los resultados obtenidos en este modelo con el compuesto UR-12460 y el antioxidante mercaptopropionilglicina sugieren que tanto el antagonismo del PAF como la captación de radicales libres pueden ser útiles en la disminución del infarto y, en general, en la mejoría de la función miocárdica durante la reperfusión y en la disminución de las arritmias. Otro importante modelo es el shock endotóxico, inducido en la rata por la administración de lipopolisacárido (LPS) de E. coli de diversos serotipos. El serotipo 0111:B4 produce un característico efecto bifásico de hipotensión arterial. El UR-12460 no inhibe la profunda e inmediata fase de hipotensión pero sí la segunda fase, más lenta y progresiva. Utilizando el serotipo 0127:B8 se ha estudiado el aumento de permeabilidad vascular en la rata. Se ha comprobado que sólo algunos órganos resultan afectados significativamente y que el aumento de permeabilidad se aprecia a partir de los 15 minutos de administrado el LPS. Por otra parte, hay una buena correlación entre la inhibición de la mortalidad inducida por PAF y la inducida por LPS en ratón, lo que refuerza la participación del PAF como mediador clave en la cascada de acontecimientos que producen la mortalidad por LPS. Este papel queda reforzado por la eficacia mostrada por el UR-12460 al atenuar las alteraciones bioquímicas y enzimáticas inducidas por LPS en rata. La inhibición del aumento del tiempo parcial de tromboplastina activada indica que el PAF puede estar también implicado en los mecanismos que conducen al síndrome de coagulación intravascular diseminada en la rata. El último modelo estudiado es el shock hipovolémico hemorrágico, en el cual la eficacia de los antagonistas probados no se ha demostrado. Es posible que su administración sólo sea beneficiosa en fases iniciales de la hemorragia, cuando el shock no se ha hecho todavía progresivo o no se han liberado otros mediadores.En resumen, puede decirse que el UR-12460 se ha mostrado activo, al menos parcialmente, en varios modelos de shock, reforzando la idea de la participación importante del PAF en estos procesos de tan diversa etiología. Sin embargo, el hecho de que este compuesto no tenga efecto sobre algunos de los marcadores del shock estudiados hace pensar que el futuro de un posible tratamiento del shock pasaría por la administración conjunta de diferentes sustancias, dirigidas cada una a los diferentes mediadores del shock que, además del PAF, intervienen en mayor o menor grado en su etiopatogenia. / Shock is a clinical situation that has been observed for centuries and whose presence has been associated to processes leading usually to death. The study of these processes has allowed to reach the conclusion that shock is a multifactorial and multietiological component syndrome. Since the cascade of events that take place during shock is very complex, it seems unlikely that acting on a single of its steps may be sufficient to block the shock state in its entirety. Nevertheless, in the last years different therapeutic approaches are being studied, among them the antagonism of platelet activating factor (PAF). The different studies carried out in this thesis intend to be a contribution to the study of the role of PAF and of its antagonists in shock. With a view to this we have performed several experimental models, both in vitro and in vivo, using UR-12460, a potent and selective PAF antagonist. The studied models are: anaphylactic shock, splachnic and coronary ischemia-reperfusion-induced shock, endotoxic shock and hemorrhagic shock. UR-12460 was found to be effective in active anaphylactic shock models, both in guinea pigs and mice. Furthermore, for a series of PAF antagonists, we have observed a good correlation between the inhibition of PAF-induced mortality and of active anaphylactic shock -induced mortality in mice, which indicates that PAF is involved in these processes. The ineffectiveness of UR-12460 in passive shock processes suggests a minor relative importance of PAF with respect to other mediators such as histamine. On the other hand, in splachnic ischemia-reperfusion (ischemia-reperfusion) shock models in the rat, UR-12460 has been shown partially effective, inhibiting the mortality and thrombocytopenia induced by shock. Another model of ischemia-reperfusion, but at myocardial level in this case, has been extensively studied in the rat. The occlusion during 6 minutes of the left coronary artery in the anesthetized rat, and subsequent reperfusion during 10 minutes, produces a number of disturbances in cardiac rhythm that are inhibited by UR-12460. This compound reduces the incidence of ventricular tachycardia during the occlusion phase as well as the onset of ventricular fibrillation in the reperfusion. This finding supports the role of PAF in the arrhythmogenesis by ischemia-reperfusion. The ischemia-reperfusion tests in isolated rat heart using the Langendorff method, combined with the subsequent incubation of the cardiac tissue with triphenyltetrazolium chloride, allow to combine in a single experiment, and under identical conditions, the macroscopic assessment of the infarcted area and the functional alterations of the heart. In this model, hearts are perfused with blood diluted with Krebs solution and are compared with hearts receiving only Krebs. The marked differences observed between the ischemic hearts that have received diluted blood and those that have not suggest that blood cells are a key factor in the genesis of infarction and in the impairment of cardiac function. On the other hand, the absence of a direct relationship between the time of ischemia-reperfusion and the severity of the infarction in the absence of blood suggests that the situation of anoxia is not a relevant factor in the genesis of infarction. There is indeed a relationship between the time of ischemia-reperfusion and the impairment of the mechanical function. The presence of blood cells may represent an amplification system of the damage caused by the anoxia. The results obtained in this model with the compound UR-12460 and the antioxidant mercaptopropionylglycine suggest that both PAF antagonism and free radical scavenging may be useful to reduce infarction and, in general, to improve the myocardial function during reperfusion and to reduce arrhythmias.Another important model is the endotoxic shock, which is induced in the rat by the administration of E. coli lipopolysaccharide (LPS) of different serotypes. Serotype 0111:B4 produces a characteristic biphasic effect of arterial hypotension. UR-12460, although not able to inhibit the steep and immediate phase of hypotension, clearly inhibits the second phase, which is slower and more progressive. The increase in vascular permeability has been studied in the rat using serotype 0127:B8. We have observed that only some organs are significantly affected and that the increase in permeability is noticeable from 15 minutes post-administration of LPS. UR-12460 has been shown to inhibit extravasation in the trachea and seminal vesicle. On the other hand, we have observed a good correlation between the inhibition of the mortality induced by PAF and that induced by LPS in the mouse, which fact reinforces the involvement of PAF as a key mediator in the cascade of events that cause the mortality by LPS. This is additionally supported by the ability shown by UR-12460 to reduce the biochemical and enzymatic alterations induced by LPS in the rat. The inhibition of the increase in the activated partial thromboplastin time shows that PAF may also be involved in the mechanisms leading to disseminated intravascular coagulation syndrome in the rat. The last shock model we have studied is the hemorrhagic hypovolemic shock, where none of the antagonists tested has proved effective. It is possible that their administration may only be beneficial in the initial stages of hemorrhage, when shock has not yet become progressive or other mediators have not yet been released. To sum up, it can be said that UR-12460 has proved effective, at least partially, in several models of shock, reinforcing the theory that PAF has a key role in these processes of such diverse etiology. However, the fact that this compound has no effect on some of the shock markers studied in this thesis brings us to think that the future of a possible treatment for shock requires the administration of different substances, directed to each one of the different mediators of shock that, in addition to PAF, are involved to a greater or lesser extent in its etiopathogenesis.

Prokaryotic and eukaryotic organisms respond to various stressors with the production of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is constitutively expressed in most adult mammalian tissues. In the present study, we have examined for the first time the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. The Xenopus hsp110 cDNA encodes an 854 amino acid protein, which shares 74% identity with mice and humans. In Xenopus A6 kidney epithelial cells hsp110 mRNA was detected constitutively and was heat inducible. Enhanced hsp110 mRNA levels were detected within 1 h, and remained elevated for at least 6 h. A similar accumulation of hsp70 mRNA was observed, but only in response to stress. Treatment of A6 cells with sodium arsenite and cadmium chloride also induced hsp110 and hsp70 mRNA accumulation. However, while ethanol treatment resulted in the accumulation of hsp70 mRNA no effect was seen for hsp110. Similarly, HSP110 and HSP70 protein increased after a 2 h heat shock and 12 h sodium arsenite treatment. The elevation in HSP110 and HSP70 protein in response to heat was detectable for up to 6 h. Recent studies with mice suggest an important role for HSP110 during development. Analysis of Xenopus embryos revealed that hsp110 mRNA was present in unfertilized eggs, indicating that it is a maternal mRNA, unlike the hsp70 message which was only detectable in response to heat shock. Heat shock-induced hsp110 mRNA accumulation was developmentally regulated, similar to hsp70, since it was not detectable until after the midblastula stage of development. Enhanced hsp110 mRNA accumulation was evident with heat shock at the blastula stage, and levels continued to increase reaching a maximum at the late tailbud stage. Message for the small heat shock protein, hsp27, was not detectable until the early tailbud stage, indicating that this hsp was not present maternally and was developmentally regulated. In situ hybridization analysis revealed that hsp110 mRNA was present in control embryos in the lens placode, spinal cord and somites, but increased upon heat shock in the anterior and posterior region, the lens placode, as well as in the somites and spinal cord. A similar distribution was observed for the hsp27 message, although it was not detectable until the early tailbud stage in control or heat-shocked embryos. The intracellular localization of HSP110 protein in response to stress was also investigated. HSP110 was detected predominantly in the cytoplasm in either a diffuse pattern or in long spindle-shaped fibres. Additionally, HSP110 was present in the nucleus. In heat shocked Xenopus A6 cells, HSP110 localized in distinct patterns surrounding the nucleus and was enhanced in the nucleus after prolonged heat stress. Sodium arsenite-treated cells displayed a similar pattern in which HSP110 localized on opposite ends of the nucleus. In contrast, in response to stress HSP30 was homogeneously distributed in the cytoplasm, moving into the nucleus only upon intense stress. This study presents, for the first time, a characterization of HSP110 in Xenopus laevis, adding to the growing knowledge of HSPs in this important model organism.

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.

Heat shock proteins (HSPs) are stress-inducible and evolutionarily conserved molecular chaperones that are involved in protein binding and translocation. As molecular chaperones, HSPs bind to denatured proteins, inhibit their aggregation, maintain their solubility, and assist in refolding. This process inhibits the formation of protein aggregates which can be lethal to the cell. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most non-native proteins. Furthermore, proteasome inhibition has been shown to induce hsp gene expression. Celastrol, a quinone methide triterpene, was shown to have an inhibitory effect on proteasome function in mammalian cells. The present study determined that celastrol induced the accumulation of ubiquitinated proteins and reduced proteasomal chymotrypsin-like activity in Xenopus laevis A6 kidney epithelial cells. In addition, incubation of A6 cells with celastrol induced the accumulation of HSP30 and HSP70 in a dose- and time-dependent manner with maximal levels of HSP accumulation occurring after 18 h of exposure. In A6 cells recovering from celastrol, the relative levels of HSP30 and HSP70 accumulation remained elevated for 18-24 h after removal of celastrol. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in celastrol-induced hsp gene expression in A6 cells, since the HSF1 inhibitor, KNK437, repressed the accumulation of HSP30 and HSP70. Exposure of A6 cells to simultaneous celastrol and mild heat shock treatment enhanced the accumulation of HSP30 and HSP70 to a greater extent than the sum of both stressors individually. Additionally, concurrent treatment of A6 cells with low concentrations of both celastrol and MG132 produced different patterns of HSP30 and HSP70 accumulation. While combined treatment with celastrol and MG132 acted synergistically on HSP30 accumulation, relative levels of HSP70 were similar to those observed with MG132 alone. Immunocytochemical analysis of celastrol- or MG132-treated A6 cells revealed HSP30 accumulation in a punctate pattern primarily in the cytoplasm with some staining in the nucleus. Also, in some cells treated with celastrol or MG132 large HSP30 staining structures were observed in the cytoplasm. Lastly, exposure of A6 cells to celastrol induced rounder cell morphology, reduced adherence and disorganization of the actin cytoskeleton. In conclusion, this study has shown that celastrol inhibited proteasome activity in amphibian cultured cells and induced HSF1-mediated expression of hsp genes.

Prokaryotic and eukaryotic organisms respond to various stressors with the production of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is constitutively expressed in most adult mammalian tissues. In the present study, we have examined for the first time the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. The Xenopus hsp110 cDNA encodes an 854 amino acid protein, which shares 74% identity with mice and humans. In Xenopus A6 kidney epithelial cells hsp110 mRNA was detected constitutively and was heat inducible. Enhanced hsp110 mRNA levels were detected within 1 h, and remained elevated for at least 6 h. A similar accumulation of hsp70 mRNA was observed, but only in response to stress. Treatment of A6 cells with sodium arsenite and cadmium chloride also induced hsp110 and hsp70 mRNA accumulation. However, while ethanol treatment resulted in the accumulation of hsp70 mRNA no effect was seen for hsp110. Similarly, HSP110 and HSP70 protein increased after a 2 h heat shock and 12 h sodium arsenite treatment. The elevation in HSP110 and HSP70 protein in response to heat was detectable for up to 6 h. Recent studies with mice suggest an important role for HSP110 during development. Analysis of Xenopus embryos revealed that hsp110 mRNA was present in unfertilized eggs, indicating that it is a maternal mRNA, unlike the hsp70 message which was only detectable in response to heat shock. Heat shock-induced hsp110 mRNA accumulation was developmentally regulated, similar to hsp70, since it was not detectable until after the midblastula stage of development. Enhanced hsp110 mRNA accumulation was evident with heat shock at the blastula stage, and levels continued to increase reaching a maximum at the late tailbud stage. Message for the small heat shock protein, hsp27, was not detectable until the early tailbud stage, indicating that this hsp was not present maternally and was developmentally regulated. In situ hybridization analysis revealed that hsp110 mRNA was present in control embryos in the lens placode, spinal cord and somites, but increased upon heat shock in the anterior and posterior region, the lens placode, as well as in the somites and spinal cord. A similar distribution was observed for the hsp27 message, although it was not detectable until the early tailbud stage in control or heat-shocked embryos. The intracellular localization of HSP110 protein in response to stress was also investigated. HSP110 was detected predominantly in the cytoplasm in either a diffuse pattern or in long spindle-shaped fibres. Additionally, HSP110 was present in the nucleus. In heat shocked Xenopus A6 cells, HSP110 localized in distinct patterns surrounding the nucleus and was enhanced in the nucleus after prolonged heat stress. Sodium arsenite-treated cells displayed a similar pattern in which HSP110 localized on opposite ends of the nucleus. In contrast, in response to stress HSP30 was homogeneously distributed in the cytoplasm, moving into the nucleus only upon intense stress. This study presents, for the first time, a characterization of HSP110 in Xenopus laevis, adding to the growing knowledge of HSPs in this important model organism.

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.