RESUMEN
Gene expression is temporally and spatially regulated by the interaction of transcription factors (TFs) and cis-regulatory elements (CREs). The uneven distribution of TF binding sites across the genome poses challenges in understanding how this distribution evolves to regulate spatio-temporal gene expression and consequent heritable phenotypic variation. In this study, chromatin accessibility profiles and gene expression profiles were collected from several species including mammals (human, mouse, bovine), fish (zebrafish and medaka), and chicken. Transcription factor binding sites clustered regions (TFCRs) at different embryonic stages were characterized to investigate regulatory evolution. The study revealed dynamic changes in TFCR distribution during embryonic development and species evolution. The synchronization between TFCR complexity and gene expression was assessed across species using RegulatoryScore. Additionally, an explainable machine learning model highlighted the importance of the distance between TFCR and promoter in the coordinated regulation of TFCRs on gene expression. Our results revealed the developmental and evolutionary dynamics of TFCRs during embryonic development from fish, chicken to mammals. These data provide valuable resources for exploring the relationship between transcriptional regulation and phenotypic differences during embryonic development.
Asunto(s)
Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Aprendizaje Automático , Factores de Transcripción , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Sitios de Unión , Humanos , Ratones , Bovinos , Oryzias/genética , Oryzias/metabolismo , Oryzias/embriología , Pollos/genética , Desarrollo Embrionario/genética , Regiones Promotoras Genéticas , Cromatina/metabolismo , Cromatina/genéticaRESUMEN
To date only four recombinant growth factors, including Filgrastim (rhG-CSF), have been approved by FDA as radiomitigators to ameliorate hematopoietic acute radiation syndrome (H-ARS). These approved agents are not stable under room-temperature, needing to be stored at 2-8 °C, and would not be feasible in a mass casualty scenario where rapid and cost-effective intervention is crucial. Delta-tocotrienol (δ-T3H), the most potent G-CSF-inducing agent among vitamin E isoforms, exhibited efficiency and selectivity on G-CSF production in comparison with TLR and STING agonists in mice. Five-dose δ-T3H was utilized as the optimal therapeutic regimen due to long-term G-CSF production and the best peripheral blood (PB) recovery of irradiated mice. Comparable with rhG-CSF, sequential administration of δ-T3H post-irradiation improved hematologic recovery and accelerated the regeneration of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow (BM) and spleen of 6.5Gy irradiated mice; and consistently enhanced repopulation of BM-HSCs. In 4.0Gy irradiated nonhuman primates, δ-T3H exhibited comparable efficacy as rhG-CSF to promote PB recovery and colony-formation of BM-HPCs. Altogether, we demonstrated that sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production, indicated δ-T3H as a promising radiomitigator for the management of H-ARS, particularly in a mass casualty scenario.
Asunto(s)
Médula Ósea , Células Madre Hematopoyéticas , Vitamina E , Animales , Ratones , Médula Ósea/patología , Médula Ósea/efectos de la radiación , Factor Estimulante de Colonias de Granulocitos/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/metabolismo , Primates , Proteínas Recombinantes/farmacología , Vitamina E/análogos & derivados , Vitamina E/uso terapéuticoRESUMEN
Ionizing radiation (IR)-induced hematopoietic injury has become a global concern in the past decade. The underlying cause of this condition is a compromised hematopoietic reserve, and this kind of hematopoietic injury could result in infection or bleeding, in addition to lethal mishaps. Therefore, developing an effective treatment for this condition is imperative. Fluacrypyrim (FAPM) is a recognized effective inhibitor of STAT3, which exhibits anti-inflammation and anti-tumor effects in hematopoietic disorders. In this context, the present study aimed to determine whether FAPM could serve as a curative agent in hematopoietic-acute radiation syndrome (H-ARS) after total body irradiation (TBI). The results revealed that the peritoneally injection of FAPM could effectively promote mice survival after lethal dose irradiation. In addition, promising recovery of peripheral blood, bone marrow (BM) cell counts, hematopoietic stem cell (HSC) cellularity, BM colony-forming ability, and HSC reconstituting ability upon FAPM treatment after sublethal dose irradiation was noted. Furthermore, FAPM could reduce IR-induced apoptosis in hematopoietic stem and progenitor cells (HSPCs) both in vitro and in vivo. Specifically, FAPM could downregulate the expressions of p53-PUMA pathway target genes, such as Puma, Bax, and Noxa. These results suggested that FAPM played a protective role in IR-induced hematopoietic damage and that the possible underlying mechanism was the modulation of apoptotic activities in HSCs.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Células Madre Hematopoyéticas , Pirimidinas , Ratones , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Acrilatos/farmacología , Apoptosis , Irradiación Corporal Total , Ratones Endogámicos C57BLRESUMEN
Differentiation therapies have been proposed to overcome the impaired cell differentiation in acute myeloid leukemia (AML). However, thus far the all-trans retinoic acid-based differentiation therapy has been the only successful modality in treating acute promyelocytic leukemia. Here, we showed that vibsanin A, a novel protein kinase C (PKC) activator, sensitized AML cells to tyrosine kinase inhibitor (TKI)-induced differentiation. Vibsanin A augmented the ability of TKIs to induce growth inhibition and G1 cell cycle arrest of AML cells. Mechanistically, PKC activation was involved in the differentiation-inducing effects of combining vibsanin A with TKIs. Moreover, we found that vibsanin A enhanced TKI-induced Lyn expression and suppression of Lyn interfered with AML cell differentiation, indicating an essential role for Lyn expression in the combination-induced differentiation. Finally, combining vibsanin A and TKIs enhanced the activation of the Raf/MEK/ERK cascade. Together, this is the first study to evaluate the synergy of vibsanin A and TKIs in AML cell differentiation. Our study lays the foundation in assessing new opportunities for the combination of vibsanin A and TKIs as a promising approach for future differentiation therapy.
Asunto(s)
Diterpenos/farmacología , Activadores de Enzimas/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Mieloides/citología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Células Mieloides/patología , Proteínas Tirosina Quinasas/metabolismoRESUMEN
In accidental irradiation situations, rapid in-field evaluation of acute radiation syndrome is critical for effective triage and timely medical treatment of irradiated individuals. A surface-enhanced Raman scattering (SERS)-based lateral flow assay was developed for the quantitative detection of C-reactive protein (CRP) as an early bio-indicator of a radiation-induced inflammatory response in nonhuman primates. Raman reporter-embedded gold-core silver-shell nanoparticles with built-in hot spots were synthesized and conjugated with a CRP detection antibody to serve as SERS tags in the lateral flow assay. The proposed SERS-based lateral flow assay can rapidly detect CRP with a limit of detection of 0.01 ng mL-1 and quantitative analysis ability. Furthermore, the assay was applied to evaluate the CRP levels in plasma samples of irradiated nonhuman primates at 0 to 80 h after exposure to sublethal (4 Gy) and lethal (8 Gy) doses of total body irradiation (n = 3 animals per group). The plasma CRP levels increase rapidly within few hours after irradiation. The CRP level peaks are observed at 12 or 24 h after irradiation, with a concentration of 201.30, 386.06 and 475.18 µg mL-1 for the 4 Gy irradiated animals and 197.14, 69.52 and 358.03 µg mL-1 for the 8 Gy irradiated animals. The results indicate the potential application of the proposed SERS-based lateral flow assay to serve as a rapid and accurate point-of-care biodosimetry assay for the quantitative detection of bio-indicators to triage irradiated individuals in the field of a radiation accident.
Asunto(s)
Proteína C-Reactiva/análisis , Inflamación/diagnóstico , Traumatismos por Radiación/diagnóstico , Espectrometría Raman , Animales , Bioensayo , Femenino , Oro , Humanos , Macaca mulatta , Masculino , Nanopartículas del Metal , PrimatesRESUMEN
Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.
Asunto(s)
ATPasas de Translocación de Protón/metabolismo , Receptores de Trombopoyetina/metabolismo , Animales , Línea Celular , Humanos , Espacio Intracelular/metabolismo , Ratones , ATPasas de Translocación de Protón Mitocondriales , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas/efectos de los fármacos , Estructura Terciaria de Proteína , Receptores de Trombopoyetina/química , Reproducibilidad de los Resultados , Trombopoyetina/farmacología , Técnicas del Sistema de Dos HíbridosRESUMEN
We conducted this study to investigate the radioprotective effects of recombinant human thrombopoietin (rhTPO) on beagle dogs irradiated with 3.0 Gy 60Co gamma rays. Fifteen healthy adult beagles were randomly assigned to a control group with alleviating care, and 5 and 10 µg/kg rhTPO treatment group. All animals received total-body irradiation using 60Co γ-ray source at a dose of 3.0 Gy (dose rate was 69.1 cGy/min). The treatment group received intramuscular injection of rhTPO 5 and 10 µg/kg at 2 h postirradiation, and the control group was administrated the same volume of normal saline. The survival rate, clinical signs, peripheral hemogram, serum biochemistry, and histopathological examination of animals in each group were assessed. Single administration of 10 µg/kg rhTPO at 2 h postirradiation promoted the recovery of multilineage hematopoiesis and improved the survival rate of beagles irradiated with 3 Gy 60Co γ rays. The administration of 10 µg/kg rhTPO alleviated fever and bleeding, reduced the requirement for supportive care, and may have mitigated multiple organ damage.
Asunto(s)
Rayos gamma , Hematopoyesis , Protectores contra Radiación , Proteínas Recombinantes , Trombopoyetina , Irradiación Corporal Total , Animales , Perros , Trombopoyetina/farmacología , Trombopoyetina/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Humanos , Protectores contra Radiación/farmacología , Protectores contra Radiación/administración & dosificación , Masculino , Radioisótopos de Cobalto , Femenino , Relación Dosis-Respuesta en la RadiaciónRESUMEN
Considering the increasing risk of nuclear attacks worldwide, the development of develop potent and safe radioprotective agents for nuclear emergencies is urgently needed. γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have demonstrated a potent radioprotective effect by inducing the production of granulocyte-colony stimulating factor (G-CSF) in vivo. However, their application is limited because of their low bioavailability. The utilization of ester prodrugs can be an effective strategy for modifying the pharmacokinetic properties of drug molecules. In this study, we initially confirmed that DT3 exhibited the most significant potential for inducing G-CSF effects among eight natural vitamin E homologs. Consequently, we designed and synthesized a series of DT3 ester and ether derivatives, leading to improved radioprotective effects. The metabolic study conducted in vitro and in vivo has identified DT3 succinate 5b as a prodrug of DT3 with an approximately seven-fold higher bioavailability compared to DT3 alone. And DT3 ether derivative 8a were relatively stable and approximately 4 times more bioavailable than DT3 prototype. Furthermore, 5b exhibited superior ability to mitigate radiation-induced pancytopenia, enhance the recovery of bone marrow hematopoietic stem and progenitor cells, and promote splenic extramedullary hematopoiesis in sublethal irradiated mice. Similarly, 8a shown potential radiation protection, but its radiation protection is less than DT3. Based on these findings, we identified 5b as a DT3 prodrug, and providing an attractive candidate for further drug development.
Asunto(s)
Sistema Hematopoyético , Profármacos , Protección Radiológica , Vitamina E/análogos & derivados , Animales , Ratones , Factor Estimulante de Colonias de Granulocitos/farmacología , Ésteres/farmacología , Éteres , Profármacos/farmacología , GranulocitosRESUMEN
Viburnum odoratissimum is a folk medicinal plant, it can dredge the meridian passage and contains mainly diterpenes, triterpenes, flavonoids, sesquiterpenes, lignans, coumarin glycosides, etc. Vibsanin-type diterpenoids are the characteristic compounds of V. odoratissimum, and are divided into eleven-membered ring, seven-membered ring, and rearrangement-type. Vibsanin B, vibsanin C and neovibsanin A are the representative compounds of the three subtypes of vibsanin-type diterpenoids respectively. V. odoratissimum has cytotoxic activity, antibacterial activity, fish piscicidal activity and activity of inhibiting the growth of plants, Cytotoxic activity is the main biological activity.
Asunto(s)
Diterpenos/aislamiento & purificación , Plantas Medicinales/química , Triterpenos/aislamiento & purificación , Viburnum/química , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Citotoxinas/química , Citotoxinas/aislamiento & purificación , Citotoxinas/farmacología , Diterpenos/química , Diterpenos/farmacología , Flavonoides/química , Flavonoides/aislamiento & purificación , Humanos , Lignanos/química , Lignanos/aislamiento & purificación , Estructura Molecular , Hojas de la Planta/química , Triterpenos/química , Triterpenos/farmacologíaRESUMEN
Nine spirostanol saponins (1-9) and seven mixtures of 25 R and 25 S spirostanol saponin isomers (10-16) were obtained from the seeds of Trigonella foenum-graecum after enzymatic hydrolysis of the furostanol saponin fraction by ß-glucosidase. Their structures were determined by NMR and MS spectroscopy. Among them, 1- 4, 6, 8, and 9 were new compounds and five, 11B, 12A, 13B, 14A, and 14B, were new structures observed from seven mixtures. In addition, the inhibitory effects of all saponins on rat platelet aggregation were evaluated.
Asunto(s)
Agregación Plaquetaria/efectos de los fármacos , Saponinas/farmacología , Espirostanos/farmacología , Trigonella/química , beta-Glucosidasa/química , Animales , Medicamentos Herbarios Chinos/farmacología , Hidrólisis , Masculino , Estructura Molecular , Extractos Vegetales/farmacología , Ratas , Ratas Wistar , Saponinas/química , Saponinas/aislamiento & purificación , Semillas/química , Espirostanos/química , Espirostanos/aislamiento & purificación , beta-Glucosidasa/metabolismoRESUMEN
Five new steroidal glycosides, timosaponin J ( 1), timosaponin K ( 2), (25 S)-karatavioside C ( 5), timosaponin L ( 6), and (25 S)-officinalisnin-I ( 8), together with eight known steroidal saponins, timosaponin E (1) ( 3), purpureagitosid ( 4), timosaponin BII ( 7), timosaponin B III ( 9), anemarrhenasaponin I ( 10), anemarrhenasaponin III ( 11), anemarrhenasaponin A (2) ( 12), and timosaponin A III ( 13), were isolated from the rhizomes of Anemarrhena asphodeloides. Their structures were elucidated on the basis of spectroscopic and chemical evidence. The aglycones of compounds 1 and 2 are new aglycones. Compounds 1- 13 were evaluated for their platelet aggregation activities, and compound 13 exhibited the strongest inhibitory effect on adenosine diphosphate (ADP)-induced platelet aggregation.
Asunto(s)
Anemarrhena/química , Glicósidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Esteroides/farmacología , Adenosina Difosfato/farmacología , Animales , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/aislamiento & purificación , Glicósidos/química , Glicósidos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Masculino , Estructura Molecular , Plantas Medicinales/química , Ratas , Ratas Wistar , Rizoma/química , Saponinas/química , Saponinas/aislamiento & purificación , Saponinas/farmacología , Esteroides/química , Esteroides/aislamiento & purificaciónRESUMEN
All-trans retinoic acid-based differentiation therapies have succeeded in the treatment of acute promyelocytic leukemia, which is a rare subtype of acute myeloid leukemia (AML). Their clinical efficacy is negligible, however, for other subtypes of AML. Here, we showed that strobilurin derivatives, a well-established class of inhibitors of mitochondrial electron transport chain (ETC) complex III, possessed differentiation-inducing activity in AML cells. Impairment of mitochondrial ETC activity was involved in the differentiation effects of strobilurin derivatives, where reactive oxygen species generation appeared unnecessary. Conversely, strobilurin derivative-mediated differentiation was triggered by pyrimidine deficiency, which resulted from the inhibition of the mitochondrial-coupled dihydroorotate dehydrogenase enzyme. Moreover, strobilurin derivative-mediated pyrimidine depletion led to the activation of the Akt/mTOR cascade, which was required for the differentiation. Our study provided evidence that strobilurin derivatives may represent a novel class of differentiation-inducing agents for the treatment of AML.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Diferenciación Celular , Inhibidores Enzimáticos/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Estrobilurinas/farmacología , Estrobilurinas/uso terapéutico , Tretinoina/farmacologíaRESUMEN
Radiation-induced intestinal injury (RIII) occurs after high doses of radiation exposure. RIII restricts the therapeutic efficacy of radiotherapy in cancer and increases morbidity and mortality in nuclear disasters. Currently, there is no approved agent for the prevention or treatment of RIII. Here, we reported that the disulfiram, an FDA-approved alcohol deterrent, prolonged the survival in mice after lethal irradiation. Pretreatment with disulfiram inhibited proliferation within 24 h after irradiation, but improved crypt regeneration at 3.5 days post-irradiation. Mechanistically, disulfiram promoted Lgr5+ intestinal stem cells (ISCs) survival and maintained their ability to regenerate intestinal epithelium after radiation. Moreover, disulfiram suppresses DNA damage accumulation, thus inhibits aberrant mitosis after radiation. Unexpectedly, disulfiram treatment did not inhibit crypt cell apoptosis 4 h after radiation and the regeneration of crypts from PUMA-deficient mice after irradiation was also promoted by disulfiram. In conclusion, our findings demonstrate that disulfiram regulates the DNA damage response and survival of ISCs through affecting the cell cycle. Given its radioprotective efficacy and decades of application in humans, disulfiram is a promising candidate to prevent RIII in cancer therapy and nuclear accident.
RESUMEN
Differentiation therapies with all-trans retinoic acid (ATRA) have been successful in treating acute promyelocytic leukemia, a rare subtype of acute myeloid leukemia (AML). However, their efficacy is limited in the case of other AML subtypes. Here, we show that the combination of ATRA with salt-inducible kinase (SIK) inhibition significantly enhances ATRA-mediated AML differentiation. SIK inhibition augmented the ability of ATRA to induce growth inhibition and G1 cell cycle arrest of AML cells. Moreover, combining ATRA and SIK inhibition synergistically activated the Akt signaling pathway but not the MAPK pathway. Pharmacological blockade of Akt activity suppressed the combination-induced differentiation, indicating an essential role for Akt in the action of the combination treatment. Taken together, our study reveals a novel role for SIK in the regulation of ATRA-mediated AML differentiation, implicating the combination of ATRA and SIK inhibition as a promising approach for future differentiation therapy.
Asunto(s)
Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Tretinoina/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Biomarcadores , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Inmunofenotipificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Sistema de Señalización de MAP Quinasas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tretinoina/uso terapéuticoRESUMEN
STAT3 protein has an important role in oncogenesis and is a promising anticancer target. Herein, we demonstrate that a novel small molecule fluacrypyrim (FAPM) inhibits the growth of leukemia cells by a predominant G1 arrest with significant decrease of the protein and mRNA levels of cyclin D1. As cyclin D1 is transcriptionally regulated by STAT3, FAPM is then shown to markedly inhibit the STAT3 phosphorylation with marginal effect on the other signal transducers and activators of transcription, and without effect on phosphoinositide-3-kinase and mitogen-activated protein kinase pathways. Further analysis shows that FAPM significantly increases the protein tyrosine phosphatases (PTPs) activity in a dose-dependent manner, and the inhibition of PTP activation by sodium pervanadate reverses FAPM-induced suppression of STAT3 tyrosine phosphorylation, indicating an important role of PTP in the action of FAPM. Finally, FAPM treatment results in selective suppression of STAT3-mediated transcriptional activity and its downstream effectors, and subsequent induction of growth arrest and apoptosis in STAT3-dependent cancer cell lines. This study therefore identifies FAPM as a potent STAT3 activation inhibitor with possible therapeutic potential against malignancies with constitutive STAT3 activation.
Asunto(s)
Acrilatos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Pirimidinas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/patología , Ciclina D/genética , Cartilla de ADN , Regulación hacia Abajo/efectos de los fármacos , Femenino , Citometría de Flujo , Fase G1/efectos de los fármacos , Células HL-60 , Humanos , Células K562 , Leucemia/metabolismo , Leucemia/patología , Fosforilación , Proteínas Tirosina Fosfatasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
It is known that the sugar chains of steroidal saponins play an important role in the biological and pharmacological activities. In order to synthesize steroidal saponins with novel sugar chains in one step for further studies on pharmacological activity, we here describe the glucosylation of steroidal saponins, and 5 compounds, timosaponin AIII (1), saponin Ta (2), saponin Tb (3), trillin (4) and cantalasaponin I (5), were converted into their glucosylated products by Toruzyme 3.0 L, a cyclodextrin glucanotransferase (CGTase). 12 glucosylated products were isolated and their structures elucidated on the basis of spectral data; they were all characterized as new compounds. The results showed that Toruzyme 3.0 L had the specific ability to add the α-D-glucopyranosyl group to the glucosyl group linked at the sugar chains of steroidal saponins, and the glucosyl group was the only acceptor. This is the first report of steroidal saponins with different degrees of glucosylation. The substrates and their glucosylated derivatives were evaluated for their cytotoxicity against HL-60 human promyelocytic leukemia cell by MTT assay. The substrates all exhibited high cytotoxicity (IC(50) < 10 µmol/L), excluding compound 5 (IC(50) > 150 µmol/L), and the cytotoxicity of most of the products showed no obvious changes compared with those of their substrates.
Asunto(s)
Glucosiltransferasas/química , Saponinas/química , Agave/química , Anemarrhena/química , Citotoxinas/farmacología , Células HL-60 , Humanos , Magnoliopsida/química , Saponinas/aislamiento & purificación , Saponinas/farmacología , Especificidad por SustratoRESUMEN
In mass casualty events involving radiation exposure, there is a substantial unmet need for identifying and developing an orally bioavailable agent that can be used to protect the hematopoietic stem cell pool and regenerate hematopoiesis after radiation injury. Dimethyl sulfoxide (DMSO), a free-radical scavenger, has shown therapeutic benefits in many preclinical and clinical studies. This study investigates the radioprotective effects of DMSO on oral administration. Single dose of oral DMSO administrated before irradiation conferred 100% survival of C57BL6/J mice receiving otherwise lethal as well as super-lethal radiation dose, with wide radioprotective time frame (from 15min to 4h). Oral DMSO not only protected radiation-induced acute hematopoietic stem and progenitor cell (HSPC) injury, but also ameliorated long-term BM suppression following irradiation in mice. Mechanistically, DMSO directly protected HSPC survival after irradiation in vitro and in vivo, whereas no radioprotective effect was seen in MLL-AF9-induced leukemia cells. Unexpectedly, DMSO treatment did not inhibit radiation-induced HSPC apoptosis, and the HSPC survival from Trp53-and PUMA-deficient mice after irradiation was also protected by DMSO. In conclusion, our findings demonstrate the radioprotective efficacy of oral DMSO. Given its oral efficacy and little toxicity, DMSO is an attractive candidate for human use in a wide variety of settings, including nuclear accidents and medical radiation.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Protectores contra Radiación , Animales , Apoptosis , Dimetilsulfóxido/farmacología , Células Madre Hematopoyéticas , Ratones , Protectores contra Radiación/farmacologíaRESUMEN
PURPOSE: Recombinant human thrombopoietin (rhTPO) has been evaluated as a therapeutic intervention for radiation-induced myelosuppression. However, the immunogenicity induced by a repeated-dosing strategy raises concerns about the therapeutic use of rhTPO. In this study, single-dose administration of rhTPO was evaluated for efficacy in the hematopoietic response and survival effect on mice and nonhuman primates exposed to total body irradiation (TBI). METHODS AND MATERIALS: Survival of lethally (9.0 Gy) irradiated C57BL/6J male mice was observed for 30 days after irradiation. Hematologic evaluations were performed on C57BL/6J male mice given a sublethal dose of radiation (6.5 Gy). Furthermore, in sublethally irradiated mice, we performed bone marrow (BM) histologic evaluation and evaluated BM-derived clonogenic activity. Next, the proportion and number of hematopoietic stem cells (HSCs) were analyzed. Competitive repopulation experiments were conducted to assess the multilineage engraftment of irradiated HSCs after BM transplantation. Flow cytometry was used to evaluate DNA damage, cell apoptosis, and cell cycle stage in HSCs after irradiation. Finally, we evaluated the efficacy of a single dose of rhTPO administered after 7 Gy TBI in male and female rhesus monkeys. RESULTS: A single administration of rhTPO 2 hours after irradiation significantly mitigated TBI-induced death in mice. rhTPO promoted multilineage hematopoietic recovery, increasing peripheral blood cell counts, BM cellularity, and BM colony-forming ability. rhTPO administration led to an accelerated recovery of BM HSC frequency and multilineage engraftment after transplantation. rhTPO treatment reduced radiation-induced DNA damage and apoptosis and promoted HSC proliferation after TBI. Notably, a single administration of rhTPO significantly promoted multilineage hematopoietic recovery and improved survival in nonhuman primates after TBI. CONCLUSIONS: These findings indicate that early intervention with a single administration of rhTPO may represent a promising and effective radiomitigative strategy for victims of radiation disasters.
Asunto(s)
Médula Ósea/efectos de la radiación , Traumatismos Experimentales por Radiación/prevención & control , Trombopoyetina/administración & dosificación , Irradiación Corporal Total/efectos adversos , Animales , Apoptosis , Recuento de Células Sanguíneas , Médula Ósea/efectos de los fármacos , Médula Ósea/lesiones , Médula Ósea/patología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/efectos de la radiación , Ciclo Celular , Daño del ADN/efectos de los fármacos , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Sistema Hematopoyético/efectos de los fármacos , Sistema Hematopoyético/lesiones , Sistema Hematopoyético/patología , Sistema Hematopoyético/efectos de la radiación , Humanos , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/administración & dosificación , Factores de TiempoRESUMEN
Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10-120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Receptores de Imidazolina/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Línea Celular , Escherichia coli/genética , Citometría de Flujo , Humanos , Hibridomas/metabolismo , Receptores de Imidazolina/genética , Receptores de Imidazolina/metabolismo , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
PURPOSE: The risk of radiation exposure is considered to have increased in recent years. For convenience and simple administration, development of an effective orally administered radioprotective agent is highly desirable. The steroid 5-androstene-3ß, 17ß-diol (5-AED) has been evaluated as both a radioprotector and a radiomitigator in mice and nonhuman primates; however, poor oral bioavailability has limited its development. A variant compound-17α-ethinyl-androst-5-ene-3ß, 17ß-diol (EAD)-exhibits significant oral bioavailability. We investigated the radioprotective effects of EAD via oral administration in mice. METHODS AND MATERIALS: Survival assays were performed in lethally (9.0-10.0 Gy) irradiated mice. Peripheral blood cell counts were monitored in lethally (9.5 Gy) or sublethally (6.5 Gy) irradiated mice. We performed histologic analysis of bone marrow (BM) and frequency and functional analysis of hematopoietic stem and progenitor cells in mice irradiated with 6.5 Gy. To investigate multilineage engraftment of irradiated hematopoietic stem cells after BM transplantation, competitive repopulation assays were conducted. Plasma granulocyte colony-stimulating factor was measured by enzyme-linked immunosorbent assay. RESULTS: Oral administration of EAD on 3 consecutive days before irradiation conferred 100% survival in mice, against otherwise 100% death, at a 9.5-Gy lethal dose of total body irradiation. EAD ameliorated radiation-induced pancytopenia at the same dose. EAD augmented BM cellular recovery and colony-forming ability, promoted hematopoietic stem and progenitor cell recovery, and expanded the pool of functionally superior hematopoietic stem cells in the BM of sublethally irradiated mice. Unlike 5-AED, EAD did not increase granulocyte colony-stimulating factor levels in mice and exhibited no therapeutic effects on hematologic recovery after irradiation; nevertheless, its radioprotective efficacy was superior to that of 5-AED. CONCLUSIONS: Our findings demonstrate the radioprotective efficacy of EAD and reveal that the 17α-ethinyl group is essential for its oral activity. Given its oral efficacy and low toxicity, EAD has potential as an optimal radioprotector for use by first responders, as well as at-risk civilian populations.