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OBJECTIVE: To investigate the feasibility of establishment of ultrasound radiomics-based models for classification of hepatic echinococcosis, so as to provide insights into precision ultrasound diagnosis of hepatic echinococcosis. METHODS: The ultrasonographic images were retrospectively collected from 200 patients with hepatic echinococcosis in Shiqu County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province in October 2014, and the regions of interest were plotted in ultrasonographic images of hepatic echinococcosis lesions. The ultrasound radiomics features of hepatic echinococcosis were extracted with 25 methods, and screened using pre-selection and the least absolute shrinkage and selection operator. Then, all ultrasonographic images were randomly assigned into the training and independent test sets according to the type of lesions at a ratio of 7:3. Machine learning models for classification of hepatic echinococcosis were created based on two classifiers, including kernel logistic regression (KLR) and medium Gaussian support vector machine (MGSVM). The receiver operating characteristic (ROC) curves were plotted, and the sensitivity, specificity and areas under the curves (AUC) of the created machine learning models for classification of hepatic echinococcosis were calculated. RESULTS: A total of 5 005 ultrasound radiomics features were extracted from 200 patients with hepatic echinococcosis using 25 methods, and 36 optimal radiomics features were screened through feature selection, based on which two machine learning models were created, including KLR and MGSVM. ROC curve analysis showed that MGS-VM presented a higher efficacy for hepatic echinococcosis classification than KLR in the training set, with a sensitivity of 0.82, a specificity of 0.78 and AUC of 0.88, while KLR presented a higher efficacy for hepatic echinococcosis classification than MGSVM in the independent test set, with a sensitivity of 0.82, a specificity of 0.72 and AUC of 0.86, respectively. CONCLUSIONS: Ultrasound radiomics-based machine learning models are feasible for hepatic echinococcosis classification.
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Equinococosis Hepática , Equinococosis , Humanos , Equinococosis Hepática/diagnóstico por imagen , Estudios de Factibilidad , Estudios Retrospectivos , UltrasonografíaRESUMEN
STUDY QUESTION: How does ectopic endometrial stromal cell (Ecto-ESC)-derived extracellular vesicular Legumain pseudogene 1 (EV-LGMNP1), a newly identified pseudogene of Legumain (LGMN), contribute to M2-phenotype macrophage polarization, and does it predict recurrence in patients with ovarian endometriosis (EMs)? SUMMARY ANSWER: EV-LGMNP1, which is abundant in Ecto-ESCs and serum from ovarian EMs, can direct macrophages towards an M2 phenotype by upregulating LGMN expression and is a promising biomarker for predicting ovarian EMs recurrence. WHAT IS KNOWN ALREADY: Extracellular vesicles (EVs) can mediate cell-to-cell crosstalk to promote disease progression via cargo molecule transport. Recently, LGMNP1, a newly identified pseudogene of LGMN, has been reported to promote cancer progression by upregulating LGMN. LGMN is a well-studied protein that can induce M2-like polarization. STUDY DESIGN, SIZE, DURATION: An in vitro study was conducted with Ecto-ESCs isolated from ectopic endometrial samples, collected from two patients with ovarian EMs (diagnosed by laparoscopy and histological analysis). A clinical retrospective cohort study of 52 ovarian EMs patients and 21 controls with available preoperative serum samples was carried out (2013-2017). The follow-up period ended either at the time of recurrence or on 31 December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ecto-ESC-derived EVs (EV/Ecto-ESCs) were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blotting. EV internalization by THP-1 cells, which are the most widely used primary human macrophages model, was detected by fluorescence labelling. After EV treatment, THP-1 cell polarization was detected by quantitative real-time PCR (qRT-PCR) and western blot analyses of CD86 (M1-related marker) and CD206 (M2-related marker). LGMNP1 mRNA expression level in EVs from both primary ectopic endometrioc stromal cells and serum was examined using qRT-PCR. Additionally, the expression of LGMN, the downstream target gene of LGMNP1, in THP-1 cells was evaluated using qRT-PCR and western blotting. Kaplan-Meier and multivariate Cox regression analyses were applied to evaluate the independent predictive factors of EMs recurrence-free survival. A novel nomogram model based on serum EV-LGMNP1 was then formulated to predict EMs recurrence. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro assays demonstrated that EV/Ecto-ESCs drove macrophages towards an M2-like phenotype. Moreover, LGMNP1 contributed to EV/Ecto-ESC-induced M2 macrophage polarization by upregulating LGMN mRNA expression levels. Clinically, serum EV-LGMNP1 was more highly expressed in recurrent EMs patients than in controls and EMs patients without recurrence. Survival analysis and our novel nomogram reconfirmed that serum EV-LGMNP1 was a novel promising and meaningful non-invasive biomarker for predicting EMs recurrence. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In vitro experiments were only performed on samples from two patients with ovarian endometriosis, and a larger sample size is needed. ESCs isolated from the eutopic endometrium of EMs and non-EMs patients should be studied in the future. Additionally, in vitro experiments should be performed using endometrial epithelium cells and further in vivo experiments, such as using mice endometriotic models to investigate whether EV/Ecto could induce M2 macrophage polarization, should be conducted. Moreover, multicentre, large-sample data are needed to validate our predictive nomogram model. WIDER IMPLICATIONS OF THE FINDINGS: Our study provides novel insights into the mechanism of M2 polarization involved in ovarian EMs progression mediated by an 'EV-shuttled pseudogene LGMNP1' mode. In addition, serum EV-LGMNP1 may serve as a novel non-invasive biomarker for predicting recurrence, providing a new therapeutic target for ovarian EMs. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by funding from the National Natural Science Foundation of China (81971361), the Natural Science Foundation of Shanghai Science and Technology (19ZR1406900), the Shanghai 'Rising Stars of Medical Talent' Youth Development Program (AB83030002019004), the Clinical Research Plan of SHDC (SHDC2020CR4087), the Shanghai Municipal Health Commission (202040498), the Research and Innovation Project of the Shanghai Municipal Education Commission (2019-01-07-00-07-E00050) and the Clinical Research Plan of SHDC (SHDC2020CR1045B). There are no competing interests to declare.
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Endometriosis , Vesículas Extracelulares , Adolescente , Animales , Biomarcadores/metabolismo , China , Cisteína Endopeptidasas , Endometriosis/patología , Endometrio/metabolismo , Femenino , Humanos , Macrófagos/metabolismo , Ratones , Seudogenes , ARN Mensajero/metabolismo , Estudios Retrospectivos , Células del Estroma/metabolismoRESUMEN
OBJECTIVE: The aim of this study was to elucidate the regulatory role of lncSNHG16 in the progression of osteosarcoma (OS) and its underlying mechanism. MATERIALS AND METHODS: Expressions of lncSNHG16, microRNA-146a-5p and NOVA1 in OS tissues and adjacent normal tissues were determined by quantitative Real-time polymerase chain reaction (qRT-PCR). Their expressions in OS cell lines were detected by qRT-PCR as well. We analyzed the relationship between lncSNHG16 expression and tumor stage, diagnosis and survival prognosis of OS patients, respectively. Cell counting kit-8 (CCK-8) and transwell experiments were conducted to explore proliferative and migratory changes of OS cells. Dual-luciferase reporter assay was used to verify the binding relationship of lncSNHG16 to microRNA-146a-5p, and microRNA-146a-5p to NOVA1. Finally, rescue experiments were performed to elucidate the regulatory effect of lncSNHG16 on the cellular behaviors of OS cells. RESULTS: LncSNHG16 was highly expressed in OS tissues and cell lines. Its expression was positively correlated with the tumor stage of OS patients. Receiver operating characteristic (ROC) curves suggested that lncSNHG16 can be used as a clinical indicator to distinguish OS patients from healthy controls. Survival analysis indicated a negative correlation between lncSNHG16 expression and survival of OS patients. Overexpression of lncSNHG16 enhanced the proliferative and migratory potentials of OS cell lines 143B and MNNG/HOS. MicroRNA-146a-5p was predicted to be the target gene of lncSNHG16, which was lowly expressed in OS tissues and cell lines. Overexpression of lncSNHG16 downregulated the expression of microRNA-146a-5p in 143B and MNNG/HOS cells. Furthermore, we verified that lncSNHG16 could bind to microRNA-146a-5p. The promotive role of lncSNHG16 in proliferative and migratory potentials of OS cells was reversed by microRNA-146a-5p. Subsequently, NOVA1 was predicted to be the target gene of microRNA-146a-5p, and was further verified by dual-luciferase reporter gene assay. Correlation analysis showed that microRNA-146a-5p expression was negatively correlated with NOVA1 expression in OS. More importantly, NOVA1 reversed the inhibitory effect of microRNA-146a-5p on the proliferative and migratory capacities of 143B and MNNG/HOS cells. CONCLUSIONS: LncSNHG16 is highly expressed in OS tissues and cell lines, participating in the development of OS by downregulating microRNA-146a-5p to upregulate NOVA1 expression.
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Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/genética , MicroARNs/genética , Osteosarcoma/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Biomarcadores de Tumor/análisis , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/mortalidad , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , MicroARNs/metabolismo , Estadificación de Neoplasias , Antígeno Ventral Neuro-Oncológico , Osteosarcoma/diagnóstico , Osteosarcoma/mortalidad , Osteosarcoma/patología , Pronóstico , ARN Largo no Codificante/análisis , Factores de TiempoRESUMEN
Objective: To establish the mouse aorta dissection (AD) model through drinking water containing ß-aminopropionitrile (BAPN). Methods: Forty 3-week-old C57B1/6J male mice were divided into four groups according to randomized block design: control, 0.2, 0.4 and 0.8 g·kg(-1)·d(-1) BAPN groups (dissolving respective dose of BAPN in the drinking water, n=10 each group). Arterial systolic blood pressure and heart rate were measured weekly in conscious, restrained mice using a noninvasive computerized tail-cuff system. Mice those died of rupture of aortic dissecting aneurysm during the study were autopsied and the aorta was examined. After 4 weeks, survived mice were sacrificed by an overdose of sodium pentobarbital and the whole aorta was harvested and analyzed. Results: The incidence of AD and the mortality of ruptured AD was 0 and 0 in control group, 30% (3/10) and 20% (2/10) in 0.2 g·kg(-1)·d(-1) BAPN group, 50% (5/10) and 40% (4/10) in 0.4 g·kg(-1)·d(-1) BAPN group, 90% (9/10) and 70% (7/10) in 0.8 g·kg(-1)·d(-1) BAPN group (both P<0.05 vs. control group). The incidence of AD and the mortality of ruptured AD increased in proportion to BAPN concentration increase. In 0.8 g·kg(-1)·d(-1) BAPN group, 7 mice died of dissecting aneurysm rupture during the experiment, among which 5 dissecting aneurysms were mainly located in the thoracic aorta and 2 dissecting aneurysms in abdominal aorta. The diameters of thoracic aorta and abdominal aorta were (1.38±0.19) and (1.23±0.13) mm in control group, (2.43±1.56) and (1.30±0.26) mm in 0.2 g·kg(-1)·d(-1) BAPN group, (2.45±1.28) and (1.30±0.31) mm in 0.4 g·kg(-1)·d(-1) BAPN group, (2.87±0.57) and (1.95±0.81) mm in 0.8 g·kg(-1)·d(-1) BAPN group (both P<0.05 vs. control group). The diameters of thoracic aorta and abdominal aorta in mice also increased in proportion with BAPN concentration increase. Furthermore, blood-filled false lumen formation and elastic fibers fragmentation were evidenced in hematoxylin-eosin stained and Vitoria blue-Sirius red stained aortic cross-sections of mice in the 0.8 g·kg(-1)·d(-1) BAPN group. Conclusion: BAPN treatment induced aortic dissection model in C57Bl/6J mice can serve as a useful wild-type mouse model for the mechanism and pharmaceutical studies of AD.
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Aneurisma de la Aorta Torácica , Disección Aórtica , Modelos Animales de Enfermedad , Aminopropionitrilo , Animales , Aorta Abdominal , Aorta Torácica , Presión Sanguínea , Frecuencia Cardíaca , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: To establish pretreatment conditions of hippuric acid (HA)and methyl-hippuric acid (MHA)in urine and HPLC conditions. METHODS: HA and MHA in urine were extracted with acetonitrile under acid condition and determinated by HPLC-DAD. The operating conditions by HPLC were C18 column (150 mm× 4.6 mm, 5 µm), methanol-0.2% acetic acid (contained 6.5 mmol/L potassium dihydrogen phosphate)(25â¶75, V/V) as mobile phase,1 ml/min as flow rate and wavelength was at 254 nm. RESULTS: The standard curves for HA, 2-MHA and 3-MHA(4-MHA)showed good linearity between 9.91~2 974.20 µg/ml(r=0.999 98), 1.91~573.60 µg/ml (r=0.999 84)and 2.00~598.65 µg/ml (r=0.999 85), respectively. The mean recoveries were 96.38%~98.01%, 83.17%~94.05 %, 103.22%~104.45%, respectively. The within-run precision were 0.50%~1.20%, 0.51%~1.59%, 0.49%~0.95%, respectively, and the between-run precision were 1.70%~3.20%, 1.30%~2.67%, 0.86%~2.74%, respectively. The detection limit of HA, 2-MHA and 3-MHA(4-MHA)were 0.18 µg/ml, 0.46 µg/ml and 0.12 µg/ml, and the low determination concentrations of the method were 0.36 µg/ml, 0.92 µg/ml and 0.24 µg/ml(1 ml urine). The urine can be kept 15 days at 4 â refrigerator without significantly loss. CONCLUSION: This method with simply pretreatment conforms to the relevant requirements of guide for establishing occupational health standards-part 5: determination methods of chemicals in biological materials. It can be used to detect HA and MHA in urine for occupational population exposure to toluene and xylene.
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Cromatografía Líquida de Alta Presión , Acetonitrilos , Hipuratos , Metilación , Tolueno , XilenosRESUMEN
Objective: To investigate the method of portable gas chromatography-mass spectroscopy (GC-MS) for the determination of common volatile organic compounds in air. Methods: The static volumetric method was used, with highly purified nitrogen gas as the diluents gas, to prepare the mixed standard gas of common volatile organic compounds with various mass concentrations. A portable GC-MS handheld probe was used for sampling and measurement, retention time and characteristic ion were used for qualitative analysis, and the full-scan mode was used for quantitative analysis. Results: The correlation coefficient of 12 volatile organic compounds determined by this method was higher than 0.999. The minimum detection mass concentration was 0.02~0.12 mg/m3, and the minimum quantitative mass concentration was 0.07~0.40 mg/m3. The relative standard deviation of precision was 4.10%~12.50%; the relative deviation of acetone, benzene, methylbenzene, and dimethylbenzene was-13.56% , 9.03% , -10.82% , and 8.67% , respectively. Conclusion: Portable GC-MS method can be used for the qualitative analysis and quantification of volatile organic compounds in occupational hazard factors and provide technical supports for identification of occupational hazard factors.
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Compuestos Orgánicos Volátiles/análisis , Benceno , Cromatografía de Gases y Espectrometría de Masas , ToluenoRESUMEN
While the nucleoporin 98-retinoic acid receptor gamma (NUP98-RARG) is the first RARG fusion protein found in acute leukemia, its roles and the molecular basis in oncogenic transformation are currently unknown. Here, we showed that homodimeric NUP98-RARG not only acquired unique nuclear localization pattern and ability of recruiting both RXRA and wild-type NUP98, but also exhibited similar transcriptional properties as RARA fusions found in acute promyelocytic leukemia (APL). Using murine bone marrow retroviral transduction/transformation assay, we further demonstrated that NUP98-RARG fusion protein had gained transformation ability of primary hematopoietic stem/progenitor cells, which was critically dependent on the C-terminal GLFG domain of NUP98 and the DNA binding domain (DBD) of RARG. In contrast to other NUP98 fusions, cells transformed by the NUP98-RARG fusion were extremely sensitive to all-trans retinoic acid (ATRA) treatment. Interestingly, while pan-RXR agonists, SR11237 and LGD1069 could specifically inhibit NUP98-RARG transformed cells, mutation of the RXR interaction domain in NUP98-RARG had little effect on its transformation, revealing that therapeutic functions of rexinoid can be independent of the direct biochemical interaction between RXR and the fusion. Together, these results indicate that deregulation of the retinoid/rexinoid signaling pathway has a major role and may represent a potential therapeutic target for NUP98-RARG-mediated transformation.
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Regulación Leucémica de la Expresión Génica , Leucemia/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Receptores de Ácido Retinoico/metabolismo , Animales , Núcleo Celular/metabolismo , Transformación Celular Neoplásica , Células HEK293 , Células HeLa , Humanos , Ratones , Microscopía Fluorescente , Unión Proteica , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae/genética , Transducción de Señal , Tretinoina/química , Receptor de Ácido Retinoico gammaRESUMEN
A novel approach to the fabrication of metal-cell-metal trilayer memory devices was demonstrated by using only two cycles of lithography and dry-etch procedures. The fabricated ultrahigh density crossbar devices can be scaled down to ≤70 nm in half-pitch without alignment issues. Depending on the different dry-etch mechanisms in transferring high and low density nanopatterns, suitable dry-etch angles and methods are studied for the transfer of high density nanopatterns. Some novel process methods have also been developed to eliminate the sidewall and other conversion obstacles for obtaining high density of uniform metallic nanopatterns. With these methods, ultrahigh density trilayer crossbar devices (~2 × 10(10) bit cm(-2)-kilobit electronic memory), which are composed of built-in practical magnetoresistive nanocells, have been achieved. This scalable process that we have developed provides the relevant industries with a cheap means to commercially fabricate three-dimensional high density metal-cell-metal nanodevices.
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The paired box domain of PAX5 was reported to fuse with the sequence of promyelocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia. In the present studies, we found, by gel shift assays, that PAX5-PML bound to a panel of PAX5-consensus sequence acts as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction analysis, PAX5-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(ΔCC) demonstrated that the coiled-coil (CC) protein interaction domain located within the PML moiety was required for PAX5-PML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP-PAX5-PML and YFP-PAX5-PML(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but not PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5-PML fusion protein has reduced intranuclear mobility compared with wild-type PAX5. Furthermore, the dimerization domain (CC) of PML was responsible for the reduced intranuclear mobility of PAX5-PML. These results indicate that the CC domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins.
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Cromosomas Humanos Par 15 , Cromosomas Humanos Par 9 , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factor de Transcripción PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Secuencia de Consenso/genética , Humanos , Ratones , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína de la Leucemia Promielocítica , Transporte de Proteínas/genética , Elementos de Respuesta/genética , Factores de Transcripción/genética , Activación Transcripcional , Transducción Genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
To investigate the mechanisms that human cytomegalovirus (HCMV) can vertically transmit from the placenta of mice to infect their offspring in the central nervous system (CNS) and cause congenital anomalies, and in order to provide basic research for preparing HCMV vaccine, we have developed a new type of mouse model of HCMV congenital CNS infection. Pure strain mice were propagated after being infected with HCMV. Then the degree of infection by HCMV to offspring was determined. The experiment shows that in the infection groups the mortality of fetal mice and the fatality of neonatal mice in one week are higher than that of the control groups (P < or = 0.05). At the same time we investigated the CNS of fetus's mice whose mothers were infected by HCMV. Our results showed: 1. The virus was successfully isolated from their cerebral cortex. 2. The signal of HCMV hybridization print was found in their nervous cell through in situ hybridization. 3. Especially human herpes virus-like particles and inclusion bodies in the plasm of nerve cell were found in the tissue of their brain under the electron microscope. This new type of mouse model of HCMV inherent CNS infection will help prepare HCMV vaccine and research HCMV congenital infection in CNS.
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Enfermedades Virales del Sistema Nervioso Central/congénito , Infecciones por Citomegalovirus/congénito , Citomegalovirus/patogenicidad , Modelos Animales de Enfermedad , Animales , Animales Recién Nacidos , Enfermedades Virales del Sistema Nervioso Central/patología , Enfermedades Virales del Sistema Nervioso Central/fisiopatología , Enfermedades Virales del Sistema Nervioso Central/virología , Corteza Cerebral/embriología , Corteza Cerebral/patología , Corteza Cerebral/virología , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/fisiopatología , Infecciones por Citomegalovirus/virología , Femenino , Humanos , Transmisión Vertical de Enfermedad Infecciosa , Masculino , Ratones , Ratones Endogámicos BALB C , Embarazo , Complicaciones Infecciosas del Embarazo/virologíaRESUMEN
AIM: To compare the pharmacokinetics of domestic and imported tablets of bendazao lysine (BL). METHODS: A single oral dose of 500 mg BL of this 2 kinds of tablets was given to 10 Chinese volunteers of Han nationality in a randomized crossover study. Plasma levels were determined with HPLC-UV method. Data were analyzed automatically by using a CAPP program on microcomputer. RESULTS: The plasma concentration-time curve was fitted to 2-compartment open model, and the major pharmacokinetic parameters of domestic and imported BL tablets were shown respectively as following: Cmax 66 +/- 16 and 65 +/- 8 mg.L-1; Tmax 0.98 +/- 0.22 and 0.98 +/- 0.21 h; T1/2 beta 6.2 +/- 1.8 and 6.2 +/- 1.7 h; AUC 335 +/- 47 and 337 +/- 58 mg.h.L-1. There was no significant difference between domestic and imported tablets. The bioavailability of the domestic vs that of the imported tablet was 99 +/- 12%. The unchanged BL in urine were about 5.4% and 5.6% respectively of the dosage in 24 h after a single oral dose. CONCLUSION: The two kinds of tablets had the same biological effects.
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Antiinflamatorios no Esteroideos/farmacocinética , Indazoles/farmacocinética , Adulto , Área Bajo la Curva , Disponibilidad Biológica , Estudios Cruzados , Humanos , Masculino , Equivalencia TerapéuticaRESUMEN
We report the molecular cloning and characterization of mouse erythrocyte protein 4.2 (P4.2). Mouse erythrocyte P4.2 is a 691-amino-acid protein with a predicted MW of 77 kDa. Northern blot analysis detected a 2.2-kb transcript in mouse reticulocytes, compared with a 2.4- to 2.5-kb transcript in human reticulocytes, which is consistent with the absence of the 30-amino-acid splicing insert in mouse erythrocyte P4.2 that is found in the human protein (isoform I). Like the human erythrocyte P4.2, mouse erythrocyte P4.2 contains regions strikingly homologous with the transglutaminase (TGase) proteins although it too most likely lacks TGase crosslinking activity. Mouse P4.2 is on average 73% identical with human erythrocyte P4.2, although regional variations exist, with greatest conservation in the regions of the molecule that contain the TGase active site, the TGase calcium-binding site, and a band 3 binding site. Hydropathy analysis reveals a protein containing a series of hydrophobic domains, similar to the situation for human P4.2 and consistent with its tight binding to the membrane, although the mouse P4.2 is missing both the strongly hydrophilic region and adjacent highly charged region that are present in the human protein, suggesting that the two proteins could differ in their physical characteristics, binding associations, or functional properties. The availability of the complete mouse erythrocyte P4.2 cDNA should help in the design of P4.2-deficient animal models (for example, ribozyme or homologous recombinant "knockout" models) that should accelerate the understanding of P4.2 function in both erythroid and non-erythroid cells.
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Proteínas Sanguíneas/genética , Membrana Eritrocítica , Proteínas de la Membrana/genética , Transglutaminasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas del Citoesqueleto , ADN Complementario , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Familia de Multigenes , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Red blood cell (RBC) protein 4.2 deficiency is often associated with a moderate nonimmune hemolytic anemia, splenomegaly, and osmotically fragile RBCs resembling, but not identical to, hereditary spherocytosis (HS). In the Japanese type of protein 4.2 deficiency (protein 4.2Nippon), the anemia is associated with a point mutation in the protein 4.2 cDNA. In this report, we describe a patient with moderate and apparently episodic nonimmune hemolytic anemia with splenomegaly, spherocytosis, osmotically fragile RBCs, reduced whole cell deformability, and abnormally dense cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proposita's RBC membrane proteins showed an 88% deficiency of protein 4.2 and a 30% deficiency of glyceraldehyde-3-phosphate dehydrogenase (band 6). Structural and molecular analyses of the proposita's protein 4.2 were normal. In contrast, limited tryptic digestion of the proposita's band 3 showed a homozygous abnormality in the cytoplasmic domain. Analysis of the pedigree disclosed six members who were heterozygotes for the band 3 structural abnormality and one member who was a normal homozygote. Direct sequence analysis of the abnormal band 3 tryptic peptide suggested that the structural abnormality resided at or near residue 40. Sequence analysis of the proposita's band 3 cDNA showed a 232G-->A mutation resulting in a 40glutamic acid-->lysine substitution (band 3Montefiore). Allele-specific oligonucleotide hybridization was used to probe for the mutation in the pedigree, showing that the proposita was homozygous, and the pedigree members who were heterozygous for the band 3 structural abnormality were also heterozygous for the band 3Montefiore mutation. The band 3Montefiore mutation was absent in 26 chromosomes from race-matched controls and in one pedigree member who did not express the band 3 structural abnormality. In coincidence with splenectomy, the proposita's anemia was largely corrected along with the disappearance of most spherocytes and considerable improvements of RBC osmotic fragility, whole cell deformability, and cell density. We conclude that this hereditary hemolytic anemia is associated with the homozygous state for band 3Montefiore (40glutamic acid-->lysine) and a decreased RBC membrane content of protein 4.2. We speculate that band 3 structural abnormalities can result in defective interactions with protein 4.2 and band 6, and in particular, that the region of band 3 containing 40glutamic acid is involved directly or indirectly in interactions with these proteins.
Asunto(s)
Anemia Hemolítica/genética , Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteínas Sanguíneas/deficiencia , Homocigoto , Adulto , Anemia Hemolítica/sangre , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Secuencia de Bases , Proteínas del Citoesqueleto , ADN/química , Deformación Eritrocítica , Membrana Eritrocítica/química , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/sangre , Humanos , Proteínas de la Membrana , Datos de Secuencia Molecular , Fragilidad Osmótica , Linaje , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Embarazo , Esferocitos/patología , Esplenomegalia , Tripsina/metabolismoRESUMEN
Erythrocyte (RBC) protein 4.2 (P4.2)-deficiency observed in Japanese individuals results in a hemolytic anemia associated with abnormally shaped (spherocytic, ovalocytic, and elliptocytic), osmotically fragile RBCs, the clinical presentation of which resembles hereditary spherocytosis (HS). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, P4.2-deficient individuals contain less than 1% of the normal membrane content of P4.2 and immunologic analysis shows that the P4.2 present exists as an equimolar doublet of 74-Kd and 72-Kd bands, in contrast to normal RBC membranes where a discrete 74-Kd band is not observed. RBC membranes from both of the biologic parents of a P4.2-deficient individual contained both the 74-Kd and the 72-Kd bands, demonstrating their heterozygosity for the P4.2 defect. The molecular basis of Japanese P4.2-deficiency was investigated by reverse transcription of total reticulocyte RNA, followed by polymerase chain reaction (PCR) amplification, subcloning, and sequencing. The complete cDNA sequence of a P4.2-deficient patient showed a single point mutation that changes codon 142 from GCT (alanine) to ACT (threonine) (Protein 4.2NIPPON). The mutation also eliminated an HgaI restriction site, therefore allowing rapid screening for the presence of the mutation. Screening of PCR-amplified genomic DNA showed that the mutation was present in the homozygous state in four (eight chromosomes) unrelated Japanese P4.2-deficient individuals and absent in 35 (70 chromosomes) P4.2-normal controls (including 15 Japanese [30 chromosomes]). The presence of the mutation was confirmed by allele-specific hybridization. The mutation occurred in an alternatively spliced exon that is present in two of four P4.2 mRNA splicing isoforms. These results demonstrate that Japanese P4.2-deficiency is closely associated with the P4.2 gene and does not arise secondarily to a defect in another membrane protein, and further suggest that the P4.2-deficiency is related to the pathogenesis of the hemolytic anemia in this variant form of recessively inherited spherocytosis.