RESUMEN
OBJECTIVE: We recently showed that host defense caerin peptides isolated from Australian frog tree were able to inhibit cervical cancer tumour cell growth in vitro. We wished to determine if radioactive isotope iodine-125 (125I) can be labeled to caerin 1.9 peptide and if this peptide is bioactive for breast cancer cells treatment. SUBJECTS AND METHODS: The biological function of caerin (1.1 and 1.9) peptides were investigated by in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The anti-cancer effect of 125I labeled caerin 1.9 was compared with unlabeled caerin 1.9 peptide. The tissue distribution of 125I labeled caerin 1.9 peptide was further studied in mice. RESULTS: In the current paper, we demonstrated that caerin peptides (1.1 and 1.9) were separately able to inhibit the viability of two breast cancer cell lines in vitro and this inhibition was more profound when these peptides were simultaneously applied. Moreover, 125I can be stably attached to caerin 1.9 peptide with high efficiency. Iodine-125 labeled caerin 1.9 inhibited breast cancer cells line MCF-7 viability more efficiently than free 125I and also than unlabeled caerin 1.9. Additionally, iodine-125 labeled caerin 1.9 in vivo imaging demonstrated that although slightly, it could be accumulated in tumor tissue. CONCLUSION: Our results from this totally original study indicated that radioactive isotope 125I labeled to caerin peptide 1.9 may be used to treat breast cancer while at the same time the response to treatment may be monitored by simultaneous imaging.
Asunto(s)
Proteínas Anfibias/química , Proteínas Anfibias/uso terapéutico , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Neoplasias de la Mama/radioterapia , Radioisótopos de Yodo/uso terapéutico , Secuencia de Aminoácidos , Proteínas Anfibias/farmacocinética , Animales , Péptidos Catiónicos Antimicrobianos/farmacocinética , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Transformación Celular Neoplásica , Femenino , Humanos , Marcaje Isotópico , Células MCF-7 , Ratones , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Distribución TisularRESUMEN
The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.
Asunto(s)
Proteínas Anfibias/farmacocinética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Animales , Autorradiografía , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Isótopos de Yodo/farmacocinética , Ratones , Ratones Transgénicos , Unión Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Hormona Liberadora de Corticotropina/genética , Distribución Tisular/efectos de los fármacos , TransfecciónRESUMEN
Naked mole-rats (Heterocephalus glaber) live in groups that are notable for their large size and caste structure, with breeding monopolized by a single female and a small number of males. Recent studies have demonstrated substantial differences between the brains of breeders and subordinates induced by changes in social standing. Corticotropin-releasing factor (CRF) receptors-which bind the hormone CRF as well as related peptides-are important regulators of stress and anxiety, and are emerging as factors affecting social behavior. We conducted autoradiographic analyses of CRF1 and CRF2 receptor binding densities in female and male naked mole-rats varying in breeding status. Both globally and in specific brain regions, CRF1 receptor densities varied with breeding status. CRF1 receptor densities were higher in subordinates across brain regions, and particularly in the piriform cortex and cortical amygdala. Sex differences were present in CRF2 receptor binding densities, as is the case in multiple vole species. CRF2 receptor densities were higher in females, both globally and in the cortical amygdala and lateral amygdalar nucleus. These results provide novel insights into the neurobiology of social hierarchy in naked mole-rats, and add to a growing body of work that links changes in the CRF system with social behavior.
Asunto(s)
Encéfalo/metabolismo , Dominación-Subordinación , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Caracteres Sexuales , Proteínas Anfibias/farmacocinética , Análisis de Varianza , Animales , Autorradiografía , Encéfalo/efectos de los fármacos , Hormona Liberadora de Corticotropina/metabolismo , Femenino , Isótopos de Yodo/farmacocinética , Masculino , Ratas Topo , Hormonas Peptídicas/farmacocinética , Unión Proteica/efectos de los fármacos , Unión Proteica/éticaRESUMEN
Ranalexin, a cationic peptide from frogs, is a potent therapeutic antimicrobial peptide (AMP). Its limited availability is an obstacle for a wider application. A high-level production of AMPs via bioengineering is possible but remains a challenging task. In the current study, we investigated the potential antibacterial properties of recombinant ranalexin, expressed in the yeast Pichia pastoris. A 78-bp DNA fragment encoding the mature ranalexin peptide with a 6-His tag on its C-terminus was designed using the preferred codon usage of P. pastoris. The gene was inserted into pPICZaA and transformed into competent cells of P. pastoris strain KM71. The yield of secretory ranalexin reached up to ~6 mg/L culture. Time-kill curve analysis of ranalexin against both Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) demonstrated a concentration- dependent rapid bactericidal activity. In checkerboard assays, the combinations of ranalexin with the established antibiotics polymyxin B or linezolid reduced the MIC additively in most tested bacteria. Time-kill assays indicated a significant synergism in E. coli and MRSA when ranalexin was used in combination with antibiotics, even at concentrations of 1/4 MIC or 1/2 MIC of ranalexin, respectively. Thus we propose that secretory ranalexin produced in P. pastoris could be a useful tool to unravel ranalexin's biological function and for use in future in vivo studies against multi- resistant bacterial infections.
Asunto(s)
Acetamidas/farmacología , Proteínas Anfibias/farmacología , Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Oxazolidinonas/farmacología , Péptidos Cíclicos/farmacología , Pichia/metabolismo , Polimixina B/farmacología , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Proteínas Anfibias/farmacocinética , Antibacterianos/farmacocinética , Bioingeniería , Sinergismo Farmacológico , Linezolid , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacocinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologíaRESUMEN
The most striking sequence difference between glucagon-like peptide-1 (GLP-1)(2) and the longer-acting GLP-1 receptor agonist, exendin-4 (Ex-4),(3) is the nine-amino acid COOH-terminal extension of Ex-4. We investigated the contribution of this extension to the survival time of Ex-4. We assessed the overall metabolism of GLP-1, Ex-4, a COOH-terminally extended GLP-1 peptide (GLP-1+Ex(31-39); GLP-Ex),(4) and a COOH-terminally truncated exendin peptide (Ex(1-30)) in anaesthetized, catheterized pigs, with focus on the extraction across the kidneys and a peripheral tissue (a hindleg, representing muscle, adipose- and connective tissue). Peptide analysis was carried out with assays against the mid-region of the peptides, whereby the role of dipeptidyl peptidase-4 (DPP-4)(5) mediated NH(2)-terminal degradation could be disregarded. The half-life of GLP-1 was significantly increased when the COOH-terminal extension of Ex-4 was added (GLP-1 4.8±3.3min; GLP-Ex 19.5±3.3min). In contrast, there was no effect of truncating Ex-4 (Ex-4 32.4±4.1min; Ex(1-30) 28.4±1.7min). Ex-4 and Ex(1-30) were cleared solely by the kidneys at rates corresponding to the glomerular filtration rate (GFR),(6) while GLP-1 and GLP-Ex were cleared by both the kidneys and peripheral tissues. Both extraction rates were, however, significantly reduced with GLP-Ex compared to GLP-1. The renal clearance rate of GLP-1 greatly exceeded GFR, while GLP-Ex was cleared at a rate resembling GFR. In conclusion, the COOH-terminal extension of Ex-4 contributes minimally to the increased survival time of Ex-4, while addition of this sequence to GLP-1 significantly reduces its clearance.
Asunto(s)
Proteínas Anfibias/sangre , Péptido 1 Similar al Glucagón/sangre , Hipoglucemiantes/sangre , Fragmentos de Péptidos/sangre , Péptidos/sangre , Ponzoñas/sangre , Secuencia de Aminoácidos , Proteínas Anfibias/síntesis química , Proteínas Anfibias/farmacocinética , Anestesia , Animales , Dipeptidil Peptidasa 4/sangre , Exenatida , Femenino , Tasa de Filtración Glomerular , Péptido 1 Similar al Glucagón/síntesis química , Péptido 1 Similar al Glucagón/farmacocinética , Semivida , Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacocinética , Lagartos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacocinética , Péptidos/síntesis química , Péptidos/farmacocinética , Estabilidad Proteica , Proteolisis , Relación Estructura-Actividad , Porcinos , Ponzoñas/síntesis química , Ponzoñas/farmacocinéticaRESUMEN
This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA- KAAGQAALGAL-NH(2) , DS 01) with phospholipid (PL) monolayers comprising (i) a lipid-rich extract of Leishmania amazonensis (LRE-La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid-air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE-La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE-La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis.