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1. To evaluate the inbreeding of yellow-feathered chickens (YFCs) and identify genes related to their unique characteristics, whole-genome re-sequencing data were applied to detect runs of homozygosity (ROH) in the genomes of 10 YFC chickens from each of 10 different YFC breeds. The number, length, distribution of ROH, and inbreeding coefficient in different YFC populations were calculated. Genomic regions with high frequency in ROH were annotated.2. In total, 25,547 ROH with an average length of 335 kb were detected, with most being <1 Mb. The domination of short ROH reflected the long breeding history of this chicken. The number, length, frequency, and distribution of ROH varied among chicken populations, and high genetic diversity was maintained.3. Numerous genes related to YFC characteristics were identified in the high-frequency ROH regions. Among these, IFNA, IFNB, IL11 RA, IL22 RA1, IFNLR1, and TRIF genes were involved in disease resistance. The AMY, G6PC, SDHB, GCNT4, and ACO genes were associated with energy material metabolism; and FABPL, AQP7, ACAA2, and RYR2 were related to meat quality and flavour. The KITLG, CREB3, RYR2, and LGR4 genes, related to pigmentation, were detected.4. This ROH-based inbreeding evaluation lays the foundation for breeding and conservation of YFC populations, and the candidate genes identified can be used for marker-assisted selection.
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Pollos , Canal Liberador de Calcio Receptor de Rianodina , Animales , Pollos/genética , Genotipo , Homocigoto , Endogamia , Polimorfismo de Nucleótido Simple , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
In this paper, a method is presented to detect the different phases of epoxy cross-linking process and the subsurface structures of SU-8 thin films by atomic force acoustic microscopy (AFAM). The AFAM imaging of SU-8 thin films was investigated under different exposure and bake conditions. Optimized conditions were obtained for the cross-linking of SU-8 thin film at the exposure does of eight laser pulses with the laser fluence 10 mJ cm-2 per pulse and the post exposure bake (PEB) time at 90 s. The subsurface structures of undeveloped SU-8 thin films were visible in the AFAM images. This method provides an effective and low-cost way for the determination of different phases of epoxy cross-linking process in nanostructured compounds, for the non-destructive testing of subsurface defects, and for the evaluation of the quality of patterned structures.
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Objective: To observe the effects of mulberry leaf polysaccharide (MLP) on insulin-like growth factor 1 (IGF-1) and insulin-like growth factor blinding protein 3 (IGFBP-3) as well as the expression of IGF-1 and IGFBP-3 mRNA in the kidney of type 1 Diabetic Nephropathy (DN) rats, and to investigate its therapeutic effects and underlying mechanisms. Methods: Type 1 DN rat model was established by intraperitoneally injecting streptozocin (STZ). SD rats were randomly divided into the control, model, insulin and MLP groups, with eight rats in each group. Rats in MLP group were given orally with MLP 200 mg/kg daily for 8 weeks and insulin group rats were given subcutaneously injection of short acting insulin 1 U daily for 8 weeks. The changes in body weight, blood and urine parameters were recorded. Extracellular matrix (ECM) was calculated. The contents of IGF-1 and IGFBP-3 in blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of IGF-1 and IGFBP-3 in the kidney were evaluated by fluorescence quantitative PCR. Results: Compared with rats in control group, blood glucose, triglyceride, total cholesterol, low density lipoprotein, very low density lipoprotein, 24 h urine protein, serum creatinine and urea nitrogen in the model group rats were significantly increased (all P<0.01), and these parameters of MLP group were significantly lower than the model group (all P<0.01). The contents of IGF-1 and IGFBP-3 in the blood serum of the model group were significantly higher than those in the control group (both P<0.001), while in the MLP group they were lower than the model group[IGF-1: (0.777±0.018) ng/ml vs (0.864±0.022) ng/ml, P<0.001; IGFBP-3: (0.759±0.016) ng/ml vs (0.846±0.021) ng/ml, P<0.001]. The mRNA expressions of IGF-1 and IGFBP-3 in the kidney of the model group were significantly higher than those in the control group (both P<0.001), while in the MLP group they were lower than in the model group (IGF-1: 1.450±0.032 vs 1.810±0.090, P<0.001; IGFBP-3: 1.684±0.018 vs 1.968±0.044, P<0.001). Compared with the model group rats, there were fewer pathological changes of kidney in MLP group rats. Conclusion: MLP has a certain therapeutic effect on DN, which may be achieved by decreasing the contents of IGF-1 and IGFBP-3 in the blood serum and down-regulating the over-expression of IGF-1 and IGFBP-3 mRNA in the kidney.
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Morus , Animales , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Nefropatías Diabéticas , Factor I del Crecimiento Similar a la Insulina , Polisacáridos , Ratas , Ratas Sprague-DawleyRESUMEN
Recently, 2-methoxyestradiol (2-ME) has been considered to be a potential anticancer agent but has not been investigated in bladder cancer. This study was conducted to clarify the role of 2-ME in bladder cancer cells. The bladder cancer cell line T-24 was treated with 2 µm 2-ME for 2 d. The T-24 cell viability, colony formation, invasion and apoptosis were observed in 2-ME-treated and control cells. The expression of hypoxia-inducible factor 1 alpha (HIF-1α) was detected using reverse transcription-polymerase chain reaction (RT-PCR). Then western blotting assay was applied to assess expressions of HIF-1α and apoptosis factors caspase-3 and Bcl-x proteins. The mRNA and protein expressions of HIF-1α in 2-ME-treated T-24 cells were remarkably lower than that of the control cells (P < 0.05). Treatment of 2-ME could significantly inhibit T-24 the cell viability, colony formation, invasion, and promote apoptosis (all P < 0.05). In addition, the protein expression of Caspase-3 was higher and that of Bcl-x protein was lower after administration of 2-ME compared to control (both P < 0.05). Collectively, we characterized the efficacy of 2-ME on bladder cancer T-24 cells as being mediated by inhibition of cell viability, colony fomation, invasion and promoting cell apoptosis, which may be achieved by suppressing HIF-1α levels. This study suggests 2-ME as a potential drug for bladder cancer therapy.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Estradiol/análogos & derivados , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , 2-Metoxiestradiol , Western Blotting , Caspasa 3/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Invasividad Neoplásica/prevención & control , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Proteína bcl-X/genéticaRESUMEN
Heavy fermions have served as prototype examples of strongly-correlated electron systems. The occurrence of unconventional superconductivity in close proximity to the electronic instabilities associated with various degrees of freedom points to an intricate relationship between superconductivity and other electronic states, which is unique but also shares some common features with high temperature superconductivity. The magnetic order in heavy fermion compounds can be continuously suppressed by tuning external parameters to a quantum critical point, and the role of quantum criticality in determining the properties of heavy fermion systems is an important unresolved issue. Here we review the recent progress of studies on Ce based heavy fermion superconductors, with an emphasis on the superconductivity emerging on the edge of magnetic and charge instabilities as well as the quantum phase transitions which occur by tuning different parameters, such as pressure, magnetic field and doping. We discuss systems where multiple quantum critical points occur and whether they can be classified in a unified manner, in particular in terms of the evolution of the Fermi surface topology.
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The nature of the pairing states of superconducting LaNiC_{2} and LaNiGa_{2} has to date remained a puzzling question. Broken time reversal symmetry has been observed in both compounds and a group theoretical analysis implies a nonunitary triplet pairing state. However, all the allowed nonunitary triplet states have nodal gap functions but most thermodynamic and NMR measurements indicate fully gapped superconductivity in LaNiC_{2}. Here we probe the gap symmetry of LaNiGa_{2} by measuring the London penetration depth, specific heat, and upper critical field. These measurements demonstrate two-gap nodeless superconductivity in LaNiGa_{2}, suggesting that this is a common feature of both compounds. These results allow us to propose a novel triplet superconducting state, where the pairing occurs between electrons of the same spin, but on different orbitals. In this case the superconducting wave function has a triplet spin component but isotropic even parity gap symmetry, yet the overall wave function remains antisymmetric under particle exchange. This model leads to a nodeless two-gap superconducting state which breaks time reversal symmetry, and therefore accounts well for the seemingly contradictory experimental results.
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The aim of this study is to investigate the role of the purinergic receptor P2X3 in the peripheral and central nervous systems during acupuncture treatment for the visceral pain of irritable bowel syndrome (IBS). A total of 24 8-day-old Sprague-Dawley (SD) neonatal male rats (SPF grade) were stimulated using colorectal distention (CRD) when the rats were awake. The modeling lasted for 2 weeks with one stimulation per day. After 6 weeks, the rats were randomly divided into three groups of eight each: (1) the normal group (NG, n = 8); (2) the model group (MG, n = 8); and (3) the model + electroacupuncture group (EA, n = 8) that received electroacupuncture at a needling depth of 5 mm at the Shangjuxu (ST37, bilateral) and Tianshu (ST25, bilateral) acupoints. The parameters of the Han's acupoint nerve stimulator (HANS) were as follows: sparse-dense wave with a frequency of 2/100 Hz, current of 2 mA, 20 min/stimulation, and one stimulation per day; the treatment was provided for seven consecutive days. At the sixth week after the treatment, the abdominal withdrawal reflex (AWR) score was determined; immunofluorescence and immunohistochemistry were used to measure the expression of the P2X3 receptor in myenteric plexus neurons, prefrontal cortex, and anterior cingulate cortex; and, a real-time PCR assay was performed to measure the expression of P2X3 messenger RNA (mRNA) in the dorsal root ganglion (DRG) and spinal cord. After stimulation with CRD, the expression levels of the P2X3 receptor in the inter-colonic myenteric plexus, DRG, spinal cord, prefrontal cortex, and anterior cingulate cortex were upregulated, and the sensitivity of the rats to IBS visceral pain was increased. Electroacupuncture (EA) could downregulate the expression of the P2X3 receptor and ease the sensitivity to visceral pain. The P2X3 receptor plays an important role in IBS visceral pain. The different levels of P2X3 in the peripheral enteric nervous system and central nervous system mediate the effects of the EA treatment of the visceral hyperalgesia of IBS.
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Sistema Nervioso Central/fisiopatología , Electroacupuntura , Síndrome del Colon Irritable/complicaciones , Síndrome del Colon Irritable/fisiopatología , Sistema Nervioso Periférico/fisiopatología , Receptores Purinérgicos P2X3 , Dolor Visceral/fisiopatología , Dolor Visceral/terapia , Puntos de Acupuntura , Animales , Animales Recién Nacidos , Regulación hacia Abajo , Sistema Nervioso Entérico/fisiopatología , Masculino , Dimensión del Dolor , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X3/biosíntesis , Receptores Purinérgicos P2X3/genética , Dolor Visceral/etiologíaRESUMEN
The aim of this study was to investigate the selection of plasma exchange (PE) parameters and the safety of children with severe ricinism. The PE parameters and heparin dosage in 7 children with severe ricinism were recorded, and changes in the patients' vital signs and coagulation function were monitored before and after PE. All patients successfully completed PE. The speed of blood flow was 50-80 mL/min, speed of exchange flow was 600-800 mL/h, and isolating rate of blood plasma was 12.5-19.05%. Transmembrane pressure was stable at approximately 100 mmHg, and venous pressure was stable at approximately 95 mmHg. The first dose of heparin was 0.39 ± 0.04 mg/kg, and the maintaining heparin dose was 0.40 ± 0.05 to 0.22 ± 0.03 mg·kg(-1)·h(-1). During the PE process, mean arterial pressure, heart rate, respiratory rate, and pulse oxygen saturation were steady. After PE, the activated partial thromboplastin time and thrombin time prolonged to 2-3 times greater than that before PE. However, no bleeding tendency was seen. For children with severe ricinism, the choice of PE to eliminate the toxin from blood, tissues, and organs was safe and effective.
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Intercambio Plasmático/métodos , Ricina/envenenamiento , Ricinus communis/envenenamiento , Coagulación Sanguínea/efectos de los fármacos , Niño , Femenino , Humanos , Masculino , Tiempo de Tromboplastina Parcial , Intercambio Plasmático/efectos adversos , Tiempo de TrombinaRESUMEN
Vitiligo is a disorder of depigmentation, for which the pathogenesis is as yet unclear. Interleukin (IL)-8 (CXCL8) is a key inflammatory chemokine. We investigated the regulation of IL-8 production in human melanocytes, and the IL-8 serum levels and skin gene expression in patients with vitiligo and in controls. Cultured melanocytes were stimulated for 24 h with tumour necrosis factor (TNF) 100 ng/mL and IL-1ß 10 ng/mL, with or without pretreatment with luteolin 50 µmol/L for 30 min, and IL-8 release was measured by ELISA. Serum cytokines were measured by a microbead array. Skin biopsies were taken from healthy subjects (n = 14) as well as from marginal lesional and nonlesional skin from patients with vitiligo (n = 15). IL-8 gene expression was evaluated by quantitative real time PCR. Both TNF and IL-1ß stimulated significant IL-8 release (P < 0.01) from melanocytes, whereas pretreatment with luteolin significantly inhibited this effect (P < 0.01). IL-8 gene expression was significantly increased in vitiligo compared with control skin (P < 0.05). IL-8 may be involved in vitiligo inflammation. Inhibition by luteolin of IL-8 release could be useful for vitiligo therapy.
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Interleucina-8/metabolismo , Luteolina/farmacología , Melanocitos/efectos de los fármacos , Vitíligo/metabolismo , Adulto , Estudios de Casos y Controles , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica , Humanos , Interleucina-1beta/farmacología , Interleucina-6/metabolismo , Masculino , Melanocitos/metabolismo , Persona de Mediana Edad , Piel/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vitíligo/tratamiento farmacológicoRESUMEN
Vitiligo is a cutaneous disorder of depigmentation, clinically characterized by well-demarcated, white macules of varying size and distribution. It can affect up to 2 percent of the population, especially younger ages. In spite of recent findings implicating genetic, immune and oxidative stress factors, the exact pathogenesis of vitiligo remains obscure. Here, we briefly discuss the prevailing theories, and offer new suggestions that could explain in part the damage of melanocyte in the vitiliginous lesions. Our emerging hypothesis is that neuropeptides released from peripheral nerve endings could synergize with new cytokines to adversely affect melanocyte function and viability. These may include corticotropin- releasing hormone (CRH) and neurotensin (NT), as well as interleukin 33 (IL-33) and thymic stromal lymphopoietin (TSLP). Such interactions could serve the basis for further research, possibly leading to new treatments.
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Vitíligo/etiología , Autoinmunidad , Hormona Liberadora de Corticotropina/fisiología , Citocinas/fisiología , Humanos , Melanocitos/fisiología , Neuropéptidos/fisiología , Estrés Oxidativo , Vitíligo/inmunología , Linfopoyetina del Estroma TímicoRESUMEN
HgCl2 is a known environemental neurotoxin, but is also used as preservative in vaccines as thimerosal containing ethyl mercury covalently linked to thiosalicylate. We recently reported that mercury choloride (HgCl(2)) can stimulate human mast cells to release vascular endothelial growth factor (VEGF), which is also vasoactive and pro-inflammatory. Here we show that thimerosal induces significant VEGF release from human leukemic cultured LAD2 mast cells (at 1 microM 326 ± 12 pg/106 cells and 335.5 ± 12 pg/106 cells at 10 microM) compared to control cells (242 ± 21 pg/106 cells, n=5, p less than 0.05); this effect is weaker than that induced by HgCl2 at 10 microM (448 ± 14 pg/106 cells) (n=3, p less than 0.05). In view of this finding, we hypothesize that the thiosalicylate component of thimerosal may have an inhibitory effect on VEGF release. Thimerosal (10 microM) added together with the peptide Substance P (SP) at 2 microM, used as a positive control, reduced VEGF release by 90 percent. Methyl thiosalicylate (1 or 10 microM) added with either SP or HgCl2 (10 microM) inhibited VEGF release by 100 percent, while sodium salicylate or ibuprofen had no effect. Pretreatment for 10 min with the flavonoid luteolin (0.1 mM) before HgCl2 or thimerosal compeletly blocked their effect. Luteolin and methyl thiosalicylate may be useful in preventing mercury-induced toxicity.
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Luteolina/farmacología , Mastocitos/efectos de los fármacos , Cloruro de Mercurio/toxicidad , Salicilatos/farmacología , Compuestos de Sulfhidrilo/farmacología , Timerosal/toxicidad , Factor A de Crecimiento Endotelial Vascular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Mastocitos/metabolismo , Sustancia P/farmacologíaRESUMEN
Objective: To investigate the clinical characteristics, treatment and prognosis of QARS1 gene related glutaminyl-tRNA synthetase deficiency. Methods: To summarize and analyze the clinical manifestations, imaging, laboratory examination, genetic variant characteristics and treatment of three patients from the Fujian Medical University Affiliated Union Hospital, the 900th Hospital of People's Liberation Army, the First Medical Center of People's Liberation Army General Hsopital carrying compound heterozygous variations in QARS1 gene with a long-term follow-up in China. A literature search was conducted using Wanfang, Weipu, China National Knowledge Infrastructure (CNKI) and Pubmed databases with the keywords "QARS", "QARS1" and "glutaminyl-tRNA Synthetase"(up to December 2019). Results: Case 1, a female 53 days of age, was admitted to the Fujian Medical University Affiliated Union Hospital for treatment because of the complaint of repetitive seizures for one month after birth and fever for one day. The seizure occurred within the first 2 hours of life with multiple forms and often had a status as persisted from hours to days. The seizures were resistant to many anti-epilepsy drugs (AED) and ketogenic diet but later controlled by clonazepam. However, she died at the age of seven years. Case 2 (younger brother of case 1), a one-hour-old boy, was hospitalized because of seizures after birth for 1 hour. Intrauterine growth retardation was discovered during late-pregnancy. The boy presented seizures and microcephaly immediately after birth, and his epilepsy was pharmacoresisitant. Case 3, an 8-month-old girl, was admitted due to recurrent convulsions for nearly two months. The girl had mild developmental retardation and hypotonia after birth. The infantile spasm was observed at her age of 6 months and disappeared under treatment with Vitamin B6, vigabatrin combined with adreno-cortico-tropic-hormone and magnesium sulfate. However, the seizure pattern turned to tonic seizures later. She was seizures free now with clobazam and zonisamide treatment. All of them manifested as a syndrome composed of severe global developmental retardation, progressive microcephaly, hypotonia from the very beginning, mild hypoproteinemia and diffuse brain atrophy. Genetic studies revealed compound heterozygous variations of QARS1 gene which were not reported previously. A review of the literature reported a total of 22 patients from 18 unrelated families all over the world. Except for 5 cases without epilepsy,all the patients shared very similar clinical manifestations as classic pentalogy. The recommended effective treatment for epilepsy has not been reported yet. Conclusions: Glutaminyl-tRNA synthetase deficiency caused by QARS1 gene variations manifested as a clinical syndrome's pentalogy, characterized by microcephaly, cerebral atrophy, intractable early-onset epileptic encephalopathy, global developmental retardation and severe muscle hypotonia.
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Aminoacil-ARNt Sintetasas/deficiencia , Aminoacil-ARNt Sintetasas/genética , China , Discapacidades del Desarrollo/genética , Epilepsia/genética , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Microcefalia/genética , Hipotonía Muscular/genética , Mutación , Embarazo , SíndromeRESUMEN
OBJECTIVE: The aim of the study was to clarify the therapeutic mechanism of Dexmedetomidine (DEX) on the chronic obstructive pulmonary disease (COPD) and its regulatory effect on long non-coding RNA (lncRNA) PACER. PATIENTS AND METHODS: Serum level of PACER in COPD patients was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic potential of PACER in COPD was assessed by plotting ROC curves. The in vivo COPD model was generated in rats by cigarette smoking exposure. Primary rat alveolar epithelial cells were isolated, purified and cultured. After overexpression of PACER in primary rat alveolar epithelial cells, proliferative and migratory abilities were assessed by cell counting kit-8 (CCK-8) and transwell assay, respectively. Subsequently, we detected changes in PACER expression, viability and migratory potentials in primary rat alveolar epithelial cells harvested from control rats, and those harvested from COPD rats and induced with either DEX or not. Rescue experiments were conducted to uncover the involvement of PP2A in PACER-regulated cell phenotypes. RESULTS: PACER was upregulated in serum of COPD patients, which was a potential biomarker for diagnosing COPD. Overexpression of PACER in primary rat alveolar epithelial cells enhanced proliferative and migratory abilities. Compared with primary rat alveolar epithelial cells harvested from control rats, proliferative and migratory abilities were stronger in those harvested from COPD rats and induced with either DEX or not. Notably, DEX induction decreased PACER expression, and proliferative and migratory abilities in primary rat alveolar epithelial cells harvested from COPD rats. Overexpression of PP2A could partially abolish the promotive effects of PACER on proliferative and migratory abilities in DEX-induced primary rat alveolar epithelial cells harvested from COPD rats. CONCLUSIONS: PACER drives the proliferative and migratory abilities of alveolar epithelial cells through activating PP2A. Dexmedetomidine is conducive to COPD treatment by downregulating PACER.
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Dexmedetomidina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , ARN Largo no Codificante/antagonistas & inhibidores , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Humanos , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , Ratas , Ratas Sprague-DawleyRESUMEN
We propose a second renormalization group method to handle the tensor-network states or models. This method dramatically reduces the truncation error of the tensor renormalization group. It allows physical quantities of classical tensor-network models or tensor-network ground states of quantum systems to be accurately and efficiently determined.
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The nacre of mollusk shells is distinguished by an exceptional mechanical efficiency which is derived essentially from its lamellar structure and frequently acts as a source of inspiration for the development of biomimetic materials. The structure and mechanical properties of nacre have been intensively investigated with a special focus on its toughening strategies; nevertheless, the fracture mechanisms, more specifically the critical stress/strain conditions for the failure of nacre, and the effects of structural orientation and hydration state remain largely unexplored. Here uniaxial compression tests were performed on nacre of both dry and hydrated states with different off-axis angles, i.e., the inclination of loading axis with respect to the lamellar structure, ranging from 0° to 90°. The mechanical properties and fracture characteristics of nacre and their dependences on the structural orientation and hydration state were elucidated in terms of mechanics behind failure. Quantitative relationships were established between the mechanical properties and off-axis angle based on different failure criteria. The competition between the fracture modes of fragmentation and shearing was quantified by comparing their respective driving force and resistance on the interfacial plane. This study may aid the understanding on the mechanical behavior of nacre and nacre-inspired synthetic materials and promote a better replication of the underlying design principles of nacre in man-made systems.
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Fenómenos Mecánicos , Nácar/química , Materiales Biomiméticos/químicaRESUMEN
Diterpenoids isolated from Labiatae family herbs have strong antitumor activities with low toxicity. In this study, Eriocalyxin B (EriB), a diterpenoid extracted from Isodon eriocalyx, was tested on human leukemia/lymphoma cells and murine leukemia models. Acute myeloid leukemia cell line Kasumi-1 was most sensitive to EriB. Significant apoptosis was observed, concomitant with Bcl-2/Bcl-XL downregulation, mitochondrial instability and caspase-3 activation. AML1-ETO oncoprotein was degraded in parallel to caspase-3 activation. EriB-mediated apoptosis was associated with NF-kappaB inactivation by preventing NF-kappaB nuclear translocation and inducing IkappaBalpha cleavage, and disturbance of MAPK pathway by downregulating ERK1/2 phosphorylation and activating AP-1. Without affecting normal hematopoietic progenitor cells proliferation, EriB was effective on primary t(8;21) leukemia blasts and caused AML1-ETO degradation. In murine t(8;21) leukemia models, EriB remarkably prolonged the survival time or decreased the xenograft tumor size. Together, EriB might be a potential treatment for t(8;21) leukemia by targeting AML1-ETO oncoprotein and activating apoptosis pathways.
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Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Diterpenos/farmacología , Leucemia Mieloide Aguda/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Proliferación Celular/efectos de los fármacos , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Diterpenos/química , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Glutatión/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Proteínas I-kappa B/metabolismo , Proteínas I-kappa B/farmacología , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteína 1 Compañera de Translocación de RUNX1 , Especies Reactivas de Oxígeno/metabolismo , Translocación Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Proteína bcl-X/metabolismoRESUMEN
SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.
Asunto(s)
Arginina/metabolismo , Ácido Aspártico/fisiología , Péptidos/metabolismo , Proteínas Quinasas , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , ARN Helicasas , Células 3T3 , Secuencia de Aminoácidos , Animales , ARN Helicasas DEAD-box , Ribonucleoproteínas Nucleares Heterogéneas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Fosforilación , Prolina/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/metabolismo , Relación Estructura-ActividadRESUMEN
Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.