RESUMO
ABSTRACT Ambystoma mexicanum is a urodele amphibian endemic to Xochimilco Lake in Mexico, it belongs to the salamander family Ambystomatidae. This species has frequently been used as model organism in developmental biology and regeneration laboratories around the world due to its broad regenerative capacities and adaptability to laboratory conditions. In this review we describe the establishment of the first colony of axolotls in Colombia to study tissue regeneration and our perspectives on the use A. mexicanum as a model organism in Colombia are discussed emphasizing its possible uses in regeneration and developmental biology.
RESUMEN Ambystoma mexicanum es un anfibio urodelo endémico del lago Xochimilco en México, perteneciente a la familia de salamandras Ambystomatidae. Esta especie se ha empleado frecuentemente como organismo modelo en laboratorios de biología del desarrollo y regeneración alrededor del mundo, dadas sus amplias capacidades regenerativas y adaptabilidad en condiciones de laboratorio. En esta revisión, se describe el establecimiento de la primera colonia de ajolotes en Colombia, para adelantar estudios de regeneración de tejidos, y se discuten las perspectivas de A. mexicanum como organismo modelo en el país, enfatizando sus posibles usos en regeneración y biología del desarrollo.
RESUMO
BACKGROUND: Hydrogen peroxide (H2 O2 ) is a key reactive oxygen species (ROS) generated during appendage regeneration among vertebrates. However, its role during tail regeneration in axolotl as redox signaling molecule is unclear. RESULTS: Treatment with exogenous H2 O2 rescues inhibitory effects of apocynin-induced growth suppression in tail blastema cells leading to cell proliferation. H2 O2 also promotes recruitment of immune cells, regulate the activation of AKT kinase and Agr2 expression during blastema formation. Additionally, ROS/H2 O2 regulates the expression and transcriptional activity of Yap1 and its target genes Ctgf and Areg. CONCLUSIONS: These results show that H2 O2 is necessary and sufficient to promote tail regeneration in axolotls. Additionally, Akt signaling and Agr2 were identified as ROS targets, suggesting that ROS/H2 O2 is likely to regulate epimorphic regeneration through these signaling pathways. In addition, ROS/H2 O2 -dependent-Yap1 activity is required during tail regeneration.
Assuntos
Ambystoma mexicanum , Peróxido de Hidrogênio , Animais , Peróxido de Hidrogênio/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regeneração/fisiologia , Cauda/fisiologiaRESUMO
In order to combat bacterial and cancer resistance, we identified peptides (pugnins) with dual antibacterial l-anticancer activity from the Boana pugnax (B. pugnax) skin transcriptome through in silico analysis. Pugnins A and B were selected owing to their high similarity to the DS4.3 peptide, which served as a template for their alignment to the B. pugnax transcriptome, as well as their function as part of a voltage-dependent potassium channel protein. The secondary peptide structure stability in aqueous medium was evaluated as well, and after interaction with the Escherichia coli (E. coli) membrane model using molecular dynamics. These pugnins were synthesized via solid-phase synthesis strategy and verified by Reverse phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry. Subsequently, their alpha-helix structure was determined by circular dichroism, after which antibacterial tests were then performed to evaluate their antimicrobial activity. Cytotoxicity tests against cancer cells also showed selectivity of pugnin A toward breast cancer (MFC7) cells, and pugnin B toward prostate cancer (PC3) cells. Alternatively, flow cytometry revealed necrotic cell damage with a major cytotoxic effect on human keratinocytes (HaCaT) control cells. Therefore, the pugnins found in the transcriptome of B. pugnax present dual antibacterial-anticancer activity with reduced selectivity to normal eukaryotic cells.
RESUMO
Peptides are naturally produced by all organisms and exhibit a wide range of physiological, immunomodulatory, and wound healing functions. Furthermore, they can provide with protection against microorganisms and tumor cells. Their multifaceted performance, high selectivity, and reduced toxicity have positioned them as effective therapeutic agents, representing a positive economic impact for pharmaceutical companies. Currently, efforts have been made to invest in the development of new peptides with antimicrobial and anticancer properties, but the poor stability of these molecules in physiological environments has triggered a bottleneck. Therefore, some tools, such as nanotechnology and in silico approaches can be applied as alternatives to try to overcome these obstacles. In silico studies provide a priori knowledge that can lead to the development of new anticancer peptides with enhanced biological activity and improved stability. This review focuses on the current status of research in peptides with dual antimicrobial-anticancer activity, including advances in computational biology using in silico analyses as a powerful tool for the study and rational design of these types of peptides.
Assuntos
Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Desenho de Fármacos , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Biologia Computacional , HumanosRESUMO
Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.
Assuntos
Proteínas de Anfíbios/metabolismo , Proteínas de Transporte/metabolismo , Proliferação de Células/fisiologia , Células-Tronco Mesenquimais/metabolismo , Salamandridae/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/classificação , Proteínas de Anfíbios/genética , Animais , Western Blotting , Células COS , Proteínas de Transporte/genética , Proliferação de Células/genética , Células Cultivadas , Chlorocebus aethiops , Cisteína/genética , Cisteína/metabolismo , Células HEK293 , Humanos , Mutação , Filogenia , Ligação Proteica , Fase S/genética , Fase S/fisiologia , Salamandridae/genética , Homologia de Sequência de AminoácidosRESUMO
UNLABELLED: The success of hepatocyte transplantation has been limited by the low efficiency of transplanted cell integration into liver parenchyma. Human fetal hepatic progenitor cells (hepatoblasts) engraft more effectively than adult hepatocytes in mouse livers. However, the signals required for their integration are not yet fully understood. We investigated the role of HGF on the migration and invasive ability of human hepatic progenitors in vitro and in vivo. Hepatoblasts were isolated from the livers of human fetuses between 10 and 12 weeks of gestation. Their invasive ability was assessed in the presence or absence of HGF. These cells were also transplanted into immunodeficient mice and analyzed by immunohistochemistry. In contrast to TNF-alpha, HGF increased the motogenesis and invasiveness of hepatoblasts, but not of human adult hepatocytes, via phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The invasive ability of human hepatoblasts correlated with the expression and secretion of matrix metalloproteinases (MMPs). Hepatoblasts stimulated with HGF prior transplantation into newborn mice migrated from the portal area into the hepatic parenchyma. CONCLUSIONS: In contrast to adult hepatocytes, hepatoblasts display invasive ability that can be modulated by HGF in vitro and in vivo.
Assuntos
Movimento Celular , Fator de Crescimento de Hepatócito/fisiologia , Hepatócitos/transplante , Transplante de Células-Tronco/métodos , Células-Tronco/fisiologia , Animais , Proliferação de Células , Feto , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Camundongos , Camundongos SCID , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Células-Tronco/citologia , Transplante HeterólogoRESUMO
Transplantation of genetically modified or unmodified hepatocytes appears to be a less invasive alternative to liver transplantation. However, clinical trials performed for the treatment of metabolic deficiencies resulted in a partial and transitory correction due to an insufficient number of engrafted and functional hepatocytes. In vitro, adult hepatocytes do not proliferate and the lack of organ donors limits their availability. Concomitantly, numerous works on hepatocyte transplantation in rodents have shown that cell engraftment was inefficient in normal livers. It is therefore necessary to explore the therapeutic potential of new cell sources such as stem cells and to develop pre-clinical models of transplantation. Foetal liver progenitor cells (hepatoblasts) are bipotent and express markers of both foetal hepatocytes and cholangiocytes. We have immortalized one clone of primate hepatoblasts using a retroviral vector expressing SV40 Large T and have characterized the cells at different population doublings (PDs). After 500 days in culture, immortalized cells remained bipotent and kept contact inhibition, in spite of numerous chromosomal rearrangements. After transplantation into athymic mice, the cells expressed hepatocyte functions but did not proliferate. We isolated, phenotypically characterized, transduced and cryopreserved early human hepatoblasts. These cells repopulate up to 7% of recipient immunodeficient mouse livers. This indicates that early progenitor cells display molecular characteristics related to proliferation and migration that allow these cells to engraft within hepatic parenchyma more efficiently than adult hepatocytes.
Assuntos
Primatas/embriologia , Transplante de Células-Tronco , Animais , Divisão Celular , Células-Tronco Hematopoéticas/citologia , Camundongos , Transplante HeterólogoRESUMO
Hepatoblasts are bipotent progenitors of both hepatocytes and cholangiocytes. The lack of stable in vitro culture systems for such cells makes it necessary to generate liver progenitor cell lines by means of immortalization. In this study, we describe the long-term behaviour of a clone of simian foetal hepatic progenitor cells immortalized by Simian virus 40 (SV40) large T-antigen (T-Ag) flanked by loxP sites. Immortalization was associated with the re-expression of telomerase activity, which decreased at late passages (population doubling 120) after more than a year in culture. This decrease was concomitant to telomere shortening and karyotypic instability. However, the chromosomes carrying the p53 gene remained intact and long-term immortalized progenitor cells maintained contact inhibition and proliferative properties. They also displayed the features of a normal bipotent phenotype. We constructed a retroviral vector expressing an inducible Cre recombinase and transferred it into the immortalized progenitors. Activation of the Cre recombinase by 4-hydroxy-tamoxifen induced SV40 T-Ag excision, leading to the death of cells expressing Cre recombinase. Immortalized progenitors at late passages stopped growing and eventually disappeared after transplantation into the livers of immunocompromised mice. These cells provide a novel model to study hepatic differentiation and carcinogenesis.
Assuntos
Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Vírus 40 dos Símios/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Linhagem Celular Transformada , Proliferação de Células , Células Cultivadas , Cromossomos de Mamíferos/metabolismo , Haplorrinos , Cariotipagem , Camundongos , Camundongos Knockout , Telomerase/metabolismo , Telômero/metabolismo , Fatores de TempoRESUMO
Transplantation of hepatocytes is a promising alternative to liver transplantation for the treatment of severe liver diseases. However, this approach is hampered by the shortage of donor organs and intrinsic limitations of adult hepatocytes. To investigate whether most of the hurdles faced with adult hepatocytes could be surmounted by the use of human fetal hepatoblasts, we have developed a method to isolate, transduce, and cryopreserve hepatoblasts from human livers at an early stage of development (11-13 weeks of gestation). Cells were characterized in vitro for expression of specific markers, and in vivo for their proliferation and differentiation potential after transplantation into athymic mice. Most of the cells (80-90%) harbored a bipotent phenotype, expressing cytokeratins 8/18, albumin, and CK19. They proliferated spontaneously in culture and were efficiently transduced by a beta-galactosidase-expressing retrovirus (90%). After transplantation, cryopreserved cells engrafted into the liver of athymic mice and proliferated, resulting in up to 10% repopulation. Engrafted cells expressed markers of differentiated adult hepatocytes including albumin, alpha1-antitrypsin, cytochrome P450 3A4, and alpha-glutathione-S-transferase. When retrovirally transduced before transplantation they expressed the transgene in vivo. In summary, early human fetal hepatoblasts engraft, proliferate, and mature in athymic mouse liver, without conditioning the donor.