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1.
Plant Physiol ; 193(1): 627-642, 2023 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-37233029

RESUMEN

Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.


Asunto(s)
Arabidopsis , Flavonoides , Plantas , Polen/genética , Arabidopsis/genética , Flavonoles , Esporas
2.
Autophagy ; 19(9): 2558-2574, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37249424

RESUMEN

Antimicrobial acroautophagy/autophagy plays a vital role in degrading intracellular pathogens or microbial molecules in host-microbe interactions. However, microbes evolved various mechanisms to hijack or modulate autophagy to escape elimination. Vector-transmitted phloem-limited bacteria, Candidatus Liberibacter (Ca. Liberibacter) species, cause Huanglongbing (HLB), one of the most catastrophic citrus diseases worldwide, yet contributions of autophagy to HLB disease proliferation remain poorly defined. Here, we report the identification of a virulence effector in "Ca. Liberibacter asiaticus" (Las), SDE3, which is highly conserved among the "Ca. Liberibacter". SDE3 expression not only promotes the disease development of HLB and canker in sweet orange (Citrus sinensis) plants but also facilitates Phytophthora and viral infections in Arabidopsis, and Nicotiana benthamiana (N. benthamiana). SDE3 directly associates with citrus cytosolic glyceraldehyde-3-phosphate dehydrogenases (CsGAPCs), which negatively regulates plant immunity. Overexpression of CsGAPCs and SDE3 significantly inhibits autophagy in citrus, Arabidopsis, and N. benthamiana. Intriguingly, SDE3 undermines autophagy-mediated immunity by the specific degradation of CsATG8 family proteins in a CsGAPC1-dependent manner. CsATG8 degradation is largely rescued by treatment with an inhibitor of the late autophagic pathway, E64d. Furthermore, ectopic expression of CsATG8s enhances Phytophthora resistance. Collectively, these results suggest that SDE3-CsGAPC interactions modulate CsATG8-mediated autophagy to enhance Las progression in citrus.Abbreviations: ACP: asian citrus psyllid; ACD2: ACCELERATED CELL DEATH 2; ATG: autophagy related; Ca. Liberibacter: Candidatus Liberibacter; CaMV: cauliflower mosaic virus; CMV: cucumber mosaic virus; Cs: Citrus sinensis; EV: empty vector; GAPC: cytosolic glyceraldehyde-3-phosphate dehydrogenase; HLB: huanglongbing; H2O2: hydrogen peroxide; Las: liberibacter asiaticus; Laf: liberibacter africanus; Lam: liberibacter americanus; Pst: Pseudomonas syringae pv. tomato; PVX: potato virus X; ROS: reactive oxygen species; SDE3: sec-delivered effector 3; TEM: transmission electron microscopy; VIVE : virus-induced virulence effector; WT: wild-type; Xcc: Xanthomonas citri subsp. citri.


Asunto(s)
Arabidopsis , Citrus , Hemípteros , Rhizobiaceae , Animales , Citrus/microbiología , Liberibacter , Peróxido de Hidrógeno , Hemípteros/fisiología , Autofagia , Enfermedades de las Plantas/microbiología
3.
Front Plant Sci ; 13: 860945, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35548310

RESUMEN

AtRsmD was recently demonstrated to be a chloroplast 16S rRNA methyltransferase (MTase) for the m2G915 modification in Arabidopsis. Here, its function of AtRsmD for chloroplast development and photosynthesis was further analyzed. The AtRsmD gene is highly expressed in green photosynthetic tissues. AtRsmD is associated with the thylakoid in chloroplasts. The atrsmd-2 mutant exhibited impaired photosynthetic efficiency in emerging leaves under normal growth conditions. A few thylakoid lamellas could be observed in the chloroplast from the atrsmd-2 mutant, and these thylakoids were loosely organized. Knockout of the AtRsmD gene had minor effects on chloroplast ribosome biogenesis and RNA loading on chloroplast ribosomes, but it reduced the amounts of chloroplast-encoded photosynthesis-related proteins in the emerging leaves, for example, D1, D2, CP43, and CP47, which reduced the accumulation of the photosynthetic complex. Nevertheless, knockout of the AtRsmD gene did not cause a general reduction in chloroplast-encoded proteins in Arabidopsis grown under normal growth conditions. Additionally, the atrsmd-2 mutant exhibited more sensitivity to lincomycin, which specifically inhibits the elongation of nascent polypeptide chains. Cold stress exacerbated the effect on chloroplast ribosome biogenesis in the atrsmd-2 mutant. All these data suggest that the AtRsmD protein plays distinct regulatory roles in chloroplast translation, which is required for chloroplast development and chloroplast function.

4.
Front Plant Sci ; 13: 878693, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35574127

RESUMEN

Reconstructing the development of sporangia in seed-free vascular plants provides crucial information about key processes enabling the production of spores that are important in the life cycle of these plants. By applying fluorescence imaging in intact tissues using dyes and confocal microscopy, this study aimed to reconstruct the key steps during the development of sporangia. Special emphasis was taken on the cell wall structures of tapetum and spore mother cells that have been challenged by microscopical documentation in the past. After staining the cell wall and cytoplasm using calcofluor white and basic fuchsin, the sporangium development of Pteris multifida was observed using confocal microscopy. The clear cell lineages from the sporangial initial cell to stalk, epidermis, inner tapetum, outer tapetum, and sporogenous cells were revealed by confocal imaging. The sporangium development improved in this work will be useful for a general understanding of fern spore formation.

5.
J Integr Plant Biol ; 64(3): 717-730, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-34958169

RESUMEN

Photoperiod/temperature-sensitive genic male sterility (P/TGMS) is widely applied for improving crop production. Previous investigations using the reversible male sterile (rvms) mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis. In this work, we isolated a restorer of rvms-2 (res3), as the male sterility of rvms-2 was rescued by res3. Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICROSPORES directly regulates UPEX1 expression, revealing a pathway for tapetum secretory function. Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3. The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3. We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation. A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Infertilidad Masculina , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fertilidad/genética , Glucanos , Infertilidad Masculina/metabolismo , Fotoperiodo , Infertilidad Vegetal/genética , Polen/metabolismo , Temperatura
6.
Front Plant Sci ; 12: 770311, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34887893

RESUMEN

Pollen coat lipids form an outer barrier to protect pollen itself and play essential roles in pollen-stigma interaction. However, the precise molecular mechanisms underlying the production, deposition, regulation, and function of pollen coat lipids during anther development remain largely elusive. In lipid metabolism, 3-ketoacyl-coenzyme A synthases (KCS) are involved in fatty acid elongation or very-long-chain fatty acid (VLCFA) synthesis. In this study, we identified six members of the Arabidopsis KCS family expressed in anther. Among them, KCS7, KCS15, and KCS21 were expressed in tapetal cells at anther stages 8-10. Further analysis demonstrated that they act downstream of male sterility 1 (MS1), a regulator of late tapetum development. The kcs7/15/21 triple mutant is fertile. Both cellular observation and lipid staining showed pollen coat lipid was decreased in kcs7/15/21 triple mutant. After landing on stigma, the wild-type pollen grains were hydrated for about 5 min while the kcs7/15/21 triple mutant pollen took about 10 min to hydrate. Pollen tube growth of the triple mutant was also delayed. These results demonstrate that the tapetum-localized KCS proteins are involved in the accumulation of pollen coat lipid and reveal the roles of tapetal-derived pollen coat lipid for pollen-stigma interaction.

7.
Front Plant Sci ; 12: 634114, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33643363

RESUMEN

The middle layer is an essential cell layer of the anther wall located between the endothecium and tapetum in Arabidopsis. Based on sectioning, the middle layer was found to be degraded at stage 7, which led to the separation of the tapetum from the anther wall. Here, we established techniques for live imaging of the anther. We created a marker line with fluorescent proteins expressed in all anther layers to study anther development. Several staining methods were used in the intact anthers to study anther cell morphology. We clarified the initiation, development, and degradation of the middle layer in Arabidopsis. This layer is initiated from both the inner and outer secondary parietal cells at stage 4, stopped cell division at stage 6, and finally degraded at stage 11. The neighboring cell layers, the epidermis, and endothecium continued cell division until stage 10, which led to a thin middle layer. The degradation of the tapetum cell wall at stage 7 lead to its isolation from the anther wall. This work presents fundamental information on the development of the middle layer, which facilitates the further investigation of anther development and plant fertility. These live imaging methods could be useful in future studies.

8.
Plant Mol Biol ; 105(6): 625-635, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33481140

RESUMEN

KEY MESSAGE: IEF, a novel plasma plasma membrane protein, is important for exine formation in Arabidopsis. Exine, an important part of pollen wall, is crucial for male fertility. The major component of exine is sporopollenin which are synthesized and secreted by tapetum. Although sporopollenin synthesis has been well studied, the transportation of it remains elusive. To understand it, we analyzed the gene expression pattern in tapetal microdissection data, and investigated the potential transporter genes that are putatively regulated by ABORTED MICROSPORES (AMS). Among these genes, we identified IMPERFECTIVE EXINE FORMATION (IEF) that is important for exine formation. Compared to the wild type, ief mutants exhibit severe male sterility and pollen abortion, suggesting IEF is crucial for pollen development and male fertility. Using both scanning and transmission electron microscopes, we showed that exine structure was not well defined in ief mutant. The transient expression of IEF-GFP driven by the 35S promoter indicated that IEF-GFP was localized in plasma membrane. Furthermore, AMS can specifically activate the expression of promoterIEF:LUC in vitro, which suggesting AMS regulates IEF for exine formation. The expression of ATP-BINDING CASSETTE TRANSPORTER G26 (AGCB26) was not affected in ief mutants. In addition, SEM and TEM data showed that the sporopollenin deposition is more defective in abcg26/ief-2 than that of in abcg26, which suggesting that IEF is involved in an independent sporopollenin transportation pathway. This work reveal a novel gene, IEF regulated by AMS that is essential for exine formation.


Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Fertilidad/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Arabidopsis/crecimiento & desarrollo , Transporte Biológico , Biopolímeros/biosíntesis , Carotenoides/metabolismo , Fertilidad/genética , Regulación de la Expresión Génica de las Plantas , Polen , Nicotiana
9.
Mol Plant ; 13(11): 1644-1653, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-32810599

RESUMEN

The outer wall of pollen and spores, namely the exine, is composed of sporopollenin, which is highly resistant to chemical reagents and enzymes. In this study, we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants. Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin. Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives, including p-hydroxybenzoate (p-BA), p-coumarate (p-CA), ferulate (FA), and lignin guaiacyl (G) units. The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis, consistent with the fact that the sporopollenin precursor originates from the tapetum. Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation. In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata, phenylpropanoid derivatives including p-BA and p-CA were also detected, but G units were not. Taken together, our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants. In addition, sporopollenin autofluorescence spectra of bryophytes, such as Physcomitrella and Haplocladium, exhibit distinct characteristics compared with those of vascular plants, indicating the diversity of sporopollenin among land plants.


Asunto(s)
Biopolímeros/química , Carotenoides/química , Fenilpropionatos/química , Plantas/química , Polen/química , Arabidopsis , Lilium , Polen/efectos de la radiación , Protectores contra Radiación
10.
Front Plant Sci ; 11: 621338, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33552112

RESUMEN

Magnesium (Mg) is an abundant and important cation in cells. Plants rely on Mg transporters to take up Mg from the soil, and then Mg is transported to anthers and other organs. Here, we showed that MGT6+/- plants display reduced fertility, while mgt6 plants are fertile. MGT6 is expressed in the anther at the early stages. Pollen mitosis and intine formation are impaired in aborted pollen grains (PGs) of MGT6+/- plants, which is similar to the defective pollen observed in mgt5 and mgt9 mutants. These results suggest that Mg deficiency leads to pollen abortion in MGT6+/- plants. Our data showed that mgt6 organs including buds develop significantly slower and mgt6 stamens accumulate a higher level of Mg, compared with wild-type (WT) and MGT6+/- plants. These results indicate that slower bud development allows mgt6 to accumulate sufficient amounts of Mg in the pollen, explaining why mgt6 is fertile. Furthermore, we found that mgt6 can restore fertility of mgt5, which has been reported to be male sterile due to defects in Mg transport from the tapetum to microspores and that an additional Mg supply can restore its fertility. Interestingly, mgt5 fertility is recovered when grown under short photoperiod conditions, which is a well-known factor regulating plant fertility. Taken together, these results demonstrate that slow development is a general mechanism to restore mgts fertility, which allows other redundant magnesium transporter (MGT) members to transport sufficient Mg for pollen formation.

11.
Plant Sci ; 277: 145-154, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30466580

RESUMEN

The sporopollenin precursors, as a general constituent of sexine, are synthesized in the tapetum and deposited on the pollen surface after transportation and processing. The polyketide synthase condenses the acyl-CoA into a hydroxyalkyl α-pyrone, which is predicted to be a component of the sporopollenin precursors. In this study, we found that the rice POLYKETIDE SYNTHASE 1 (OsPKS1) was the orthologue of Arabidopsis POLYKETIDE SYNTHASE A/LESS ADHESIVE POLLEN 6 (PKSA/LAP6) through sequence alignment. The OsPKS1 knockout mutants obtained by Crispr-Cas9-mediated editing exhibited a complete male sterile phenotype. Cytological observations revealed that abnormal bacula deposition and ubisch body structures for sexine formation led to pollen rupture in ospks1. The expression analysis showed that the OsPKS1 was highly expressed in tapetal cells and anther locules from stage 9 to stage 11 during anther development in rice. Subcellular localization demonstrated that the OsPKS1 protein was preferentially localized to the ER. The genomic sequence of OsPKS1 driven by the PKSA/LAP6 promoter restored the sexine pattern of Arabidopsis pksa/lap6. These results indicated that OsPKS1 is required for sexine layer formation in rice and functionally conserved in the sporopollenin synthesis pathway.


Asunto(s)
Arabidopsis/metabolismo , Oryza/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Oryza/fisiología , Proteínas de Plantas/metabolismo
12.
Sci Signal ; 11(547)2018 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-30206136

RESUMEN

The Hippo signaling pathway regulates organ size and plays critical roles in maintaining tissue growth, homeostasis, and regeneration. Dysregulated in a wide spectrum of cancers, in mammals, this pathway is regulated by two key effectors, YAP and TAZ, that may functionally overlap. We found that TAZ promoted liver inflammation and tumor development. The expression of TAZ, but not YAP, in human liver tumors positively correlated with the expression of proinflammatory cytokines. Hyperactivated TAZ induced substantial myeloid cell infiltration into the liver and the secretion of proinflammatory cytokines through a TEAD-dependent mechanism. Furthermore, tumors with hyperactivated YAP and TAZ had distinct transcriptional signatures, which included the increased expression of inflammatory cytokines in TAZ-driven tumors. Our study elucidated a previously uncharacterized link between TAZ activity and inflammatory responses that influence tumor development in the liver.


Asunto(s)
Proteínas de Unión al ADN/genética , Inflamación/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/genética , Hígado/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción/genética , Animales , Proteínas de Ciclo Celular , Citocinas/genética , Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Vía de Señalización Hippo , Humanos , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones Endogámicos C57BL , Mutación , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genética , Factores de Transcripción de Dominio TEA , Transactivadores , Factores de Transcripción/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Trasplante Heterólogo
13.
Nat Biotechnol ; 31(8): 753-8, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23831709

RESUMEN

By enabling the simultaneous engagement of two distinct targets, bispecific antibodies broaden the potential utility of antibody-based therapies. However, bispecific-antibody design and production remain challenging, owing to the need to incorporate two distinct heavy and light chain pairs while maintaining natural nonimmunogenic antibody architecture. Here we present a bispecific-antibody production strategy that relies on co-culture of two bacterial strains, each expressing a half-antibody. Using this approach, we produce 28 unique bispecific antibodies. A bispecific antibody against the receptor tyrosine kinases MET and EGFR binds both targets monovalently, inhibits their signaling, and suppresses MET and EGFR-driven cell and tumor growth. Our strategy allows rapid generation of bispecific antibodies from any two existing antibodies and yields milligram to gram quantities of bispecific antibodies sufficient for a wide range of discovery and preclinical applications.


Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Técnicas de Cocultivo , Receptores ErbB/inmunología , Neoplasias/terapia , Proteínas Proto-Oncogénicas c-met/inmunología , Anticuerpos Biespecíficos/inmunología , Especificidad de Anticuerpos , Bacterias/inmunología , Bacterias/metabolismo , Línea Celular Tumoral , Receptores ErbB/genética , Regulación Bacteriana de la Expresión Génica/inmunología , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Neoplasias/inmunología , Neoplasias/patología , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas c-met/genética
14.
Proc Natl Acad Sci U S A ; 110(32): E2987-96, 2013 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-23882082

RESUMEN

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Homología de Secuencia de Aminoácido
15.
Cancer Res ; 72(1): 210-9, 2012 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-22084396

RESUMEN

Combinations of MAP/ERK kinase (MEK) and phosphoinositide 3-kinase (PI3K) inhibitors have shown promise in preclinical cancer models, leading to the initiation of clinical trials cotargeting these two key cancer signaling pathways. GDC-0973, a novel selective MEK inhibitor, and GDC-0941, a class I PI3K inhibitor, are in early stage clinical trials as both single agents and in combination. The discovery of these selective inhibitors has allowed investigation into the precise effects of combining inhibitors of two major signaling branches downstream of RAS. Here, we investigated multiple biomarkers in the mitogen-activated protein kinase (MAPK) and PI3K pathway to search for points of convergence that explain the increased apoptosis seen in combination. Using washout studies in vitro and alternate dosing schedules in mice, we showed that intermittent inhibition of the PI3K and MAPK pathway is sufficient for efficacy in BRAF and KRAS mutant cancer cells. The combination of GDC-0973 with the PI3K inhibitor GDC-0941 resulted in combination efficacy in vitro and in vivo via induction of biomarkers associated with apoptosis, including Bcl-2 family proapoptotic regulators. Therefore, these data suggest that continuous exposure of MEK and PI3K inhibitors in combination is not required for efficacy in preclinical cancer models and that sustained effects on downstream apoptosis biomarkers can be observed in response to intermittent dosing.


Asunto(s)
Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Indazoles/administración & dosificación , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/administración & dosificación , Sulfonamidas/administración & dosificación , Animales , Antineoplásicos/farmacología , Western Blotting , Línea Celular , Humanos , Indazoles/farmacología , Ratones , Polimorfismo de Nucleótido Simple , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacología
16.
Cell Signal ; 23(1): 201-12, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20837138

RESUMEN

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.


Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor EphA2/metabolismo , Línea Celular Tumoral , Efrina-A1/farmacología , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Transducción de Señal , Serina-Treonina Quinasas TOR , Proteínas Activadoras de ras GTPasa/metabolismo
18.
J Integr Plant Biol ; 52(3): 254-64, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20377686

RESUMEN

The cytological events, including nuclear fusion, digestion of male organelles and rebuilding of the plasmalemma and cell wall, during zygote formation of the fern Ceratopteris thalictroides (L.) Brongn. are described based on the observations of transmission electron microscopy. When the spermatozoid enters the egg and contacts the cytoplasm, the male chromatin relaxes continually. The microtubular ribbon (MTr) is separated from the male nucleus and then an envelope reappears around the male nucleus. During nuclear fusion, the egg nucleus becomes highly irregular and extends some nuclear protrusions. It is proposed that the protrusions fuse with the male nucleus actively. After nuclear fusion the irregular zygotic nucleus contracts gradually. It becomes spherical before the zygote divides. The male chromatin is identifiable as fibrous structure in the zygotic nucleus in the beginning, but it gradually becomes diffused completely. The male organelles, including the MTr, multilayered structure, flagella and the male mitochondria are finally digested in the zygotic cytoplasm. Finally a new plasmalemma and cell wall are formed outside the protoplast. The organelles in the zygote are rearranged, which produces a horizontal polarity zygote. The zygote divides with an oblique-vertical cell plate facing the apical notch of the gametophyte.


Asunto(s)
Helechos/citología , Helechos/fisiología , Fertilización/fisiología , Cigoto/citología , Núcleo Celular/ultraestructura , Pared Celular/ultraestructura , Células Germinativas de las Plantas/citología , Células Germinativas de las Plantas/ultraestructura , Fusión de Membrana , Factores de Tiempo
19.
Biochem J ; 422(3): 433-42, 2009 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-19552627

RESUMEN

Receptor tyrosine kinases of the Eph family become tyrosine phosphorylated and initiate signalling events upon binding of their ligands, the ephrins. Eph receptors such as EphA2 and EphB4 are highly expressed but poorly tyrosine phosphorylated in many types of cancer cells, suggesting a limited interaction with ephrin ligands. Nevertheless, decreasing the expression of these receptors affects the malignant properties of cancer cells, suggesting that Eph receptors may influence cancer cells independently of ephrin stimulation. Ligand-independent activities of Eph receptors in cancer, however, have not been demonstrated. By using siRNA (small interfering RNA) to downregulate EphB4 in MCF7 and MDA-MB-435 cancer cells, we found that EphB4 inhibits integrin-mediated cell substrate adhesion, spreading and migration, and reduces beta1-integrin protein levels. Low expression of the EphB4 preferred ligand, ephrin-B2, and minimal contact between cells in these assays suggest that cell contact-dependent stimulation of EphB4 by the transmembrane ephrin-B2 ligand does not play a role in these effects. Indeed, inhibitors of ephrin-B2 binding to endogenous EphB4 did not influence cell substrate adhesion. Increasing EphB4 expression by transient transfection inhibited cell substrate adhesion, and this effect was also independent of ephrin stimulation because it was not affected by single amino acid mutations in EphB4 that impair ephrin binding. The overexpressed EphB4 was tyrosine phosphorylated, and we found that EphB4 kinase activity is important for inhibition of integrin-mediated adhesion, although several EphB4 tyrosine phosphorylation sites are dispensable. These findings demonstrate that EphB4 can affect cancer cell behaviour in an ephrin-independent manner.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Receptor EphB4/metabolismo , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Ensayo de Inmunoadsorción Enzimática , Efrina-B2/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Mutación , Unión Proteica/genética , ARN Interferente Pequeño , Receptor EphB4/genética
20.
J Integr Plant Biol ; 51(3): 243-50, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19261067

RESUMEN

The ultrastructure of the mature egg and fertilization in the fern Ceratopteris thalictroides (L.) Brongn. were observed by transmission electron microscopy. The results revealed that the mature egg possesses an obvious egg membrane at the periphery of the egg. Furthermore, a fertilization pore was identified in the upper egg membrane of the mature egg. The structure of the pore is described for the first time. The fertilization experiment indicated that spermatozoids crowd into the cavity above the egg through the neck canal of the archegonium; however, only one of these can penetrate into the egg through the fertilization pore. Immediately on penetration of the spermatozoid, the egg begins to shrink. The volume of the fertilized egg decreases to almost one-half that of the unfertilized egg. As a result, the protoplasm of the fertilized egg becomes dense and opaque, which may lead to a situation where the organelles of both the egg and the fertilizing spermatozoid become indistinguishable. Simultaneously, abundant vesicles containing concentric membranes or opaque materials appear near the fertilization pore in the cytoplasm of the fertilized egg. These vesicles are considered to act as a barrier that prevents polyspermy. The present study provides a new insight into the ultrastructure of the mature egg and the cytological mechanism of fertilization in ferns.


Asunto(s)
Helechos/ultraestructura , Células Germinativas/ultraestructura , Reproducción
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