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Objective: To investigate the inplementation of cardiovascular surgery for congenital heart disease (CHD) in China. Methods: A cross-sectional study was carried out. The CHD cardiovascular surgery data collected by the Chinese Society of Extracorporeal Circulation from 2017 to 2021 in 31 provinces (autonomous regions/municipalities) of China were retrospectively reviewed, the implementation of CHD cardiovascular surgery in different provinces, regions, general/specialized hospitals, and different age groups (whether≤18 years old) were summarized, and the correlation analysis between the number of surgeries carried out in each province/region and the gross regional product and the number of the regional population was performed. Results: Between 2017 and 2021, the annual volume of CHD cardiovascular surgery was 77 120, 77 634, 81 161, 62 663 and 71 492, respectively, showing a decreasing trend. Meanwhile, the proportion of CHD patients aged≤18 years who underwent cardiovascular surgery also showed a downward trend, from 79.8% (61 557/77 120) in 2017 to 58.6% (41 871/71 492) in 2021 (P=0.027). The number of surgical cases varied greatly among different provinces, including 4 provinces with≥5 000 cases and 9 provinces with 2 000-5 000 cases. In the five years, the number of CHD cardiovascular surgeries in Central and East China was the largest, accounting for 41.1%-45.5% of the total surgical cases. The proportion of CHD surgery cases≤18 years old was the highest in Southwest China (69.7%-87.4%) and the lowest in Northeast China (28.2%-68.9%). Except for 2021, the number of cases carried out by each region between 2017 and 2020 was correlated with the gross regional product (r=0.929, 0.929, 0.893 and 0.964, respectively, all P<0.05) and the population (r=0.821, 0.893, 0.821 and 0.857, respectively, all P<0.05). Hospitals that performed more than 100 operations (20.5%±1.2% of the total number of hospitals) completed 86.2%±1.2% of the total number of operations in China during the 5-year period. In 2017 and 2021, the number of CHD cardiovascular surgeries preformed in children's/women's and children's specialized hospitals accounted for 24.3% (18 772/77 120) and 23.8% (17 012/71 492) of the total number of cases in China, respectively. Conclusions: From 2017 to 2021, the number of cardiovascular surgery for CHD decreases slightly, but the proportion of surgery for adult CHD patients increases significantly.There is a strong correlation between the number of CHD operations in each region and their economic development status. The scale of CHD cardiovascular surgery performed in children's hospitals/women's and children's hospitals accounts for about a quarter of the total volume in China.
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Cardiopatías Congénitas , Humanos , Cardiopatías Congénitas/cirugía , China , Encuestas y Cuestionarios , Procedimientos Quirúrgicos Cardiovasculares/tendencias , Adolescente , Niño , Procedimientos Quirúrgicos CardíacosRESUMEN
Objective: To explore risk factors associated with in-hospital mortality in patients requiring extracorporeal membrane oxygenation (ECMO) in the perioperative period of heart transplantation. Methods: The data of ECMO cases in the perioperative period of heart transplantation from the Chinese Society of Extracorporeal Life Support (CSECLS) between January 2017 and December 2021 were retrospectively analyzed. These patients were divided into the survival group and non-survival group according to their outcomes at discharge. The demographics, indications and complications of ECMO between the two groups were compared, and the related risk factors of poor prognosis were analyzed. Results: A total of 77 patients were included in the study, including 67 males and 10 females, with a median age [M(Q1, Q3)] of 48 (36, 59) years. Sixty-three patients (81.8%) were successfully withdrawn from the ECMO and 46 patients (59.7%) survived to discharge. The median ECMO time was 139 (92, 253) hours. Compared with the survival group, the non-survival group (n=31) had more patients with chronic kidney disease before surgery [22.6% (7/31) vs 4.3% (2/46), P=0.034], and a higher proportion of continuous renal replacement therapy (CRRT) during ECMO [74.2% (23/31) vs 50.0% (23/46), P=0.034]. Moreover, the non-survival group had longer duration of extracorporeal circulation [262 (195, 312) vs 201 (155, 261) min, P=0.056] and higher lactate value in the first 24 hours of ECMO support [2.7 (2.1, 4.7) vs 2.3 (1.4, 3.8) mmol/L, P=0.060], but the differences were not statistically significant. Multivariate logistic regression analysis showed that perioperative application of CRRT was an independent risk factor for poor prognosis in ECMO patients during heart transplantation (OR=19.345, 95%CI: 1.209-309.440, P=0.036). Conclusion: CRRT treatment during ECMO is a risk factor for in-hospital mortality in patients undergoing heart transplantation.
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Oxigenación por Membrana Extracorpórea , Trasplante de Corazón , Femenino , Masculino , Humanos , Mortalidad Hospitalaria , Estudios Retrospectivos , Periodo Perioperatorio , Ácido Láctico , Factores de RiesgoRESUMEN
Objective: To Summarize the experience of pump-controlled retrograde trial off (PCRTO) in the process of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) withdrawal in adult patients. Methods: Adult patients who received ECMO assistance in Intensive Care Unit for Cardiac Surgery from March to July 2019 were collected. According to our strategies, PCRTO was used if the patients can wean from VA-ECMO and hemodynamic indexes were recorded during the process. The statistics data was collected, including the 48 hours survival rate, ECMO re-assistance rate, thrombus complications, Intensive Care Unit (ICU) stay time and hospital stay time after weaning from VA-ECMO. The patients who failed in the test were continued to be assisted by ECMO. Results: There were 46 patients assisted by VA-ECMO in our center. In total, 21 adults who met the offline test standard underwent 26 PCRTOs, including 10 male adults (47.6%), with an age of 65 (55, 68) years old. Eighteen adults passed the withdrawal test. No new thrombus was found in the arteriovenous ultrasound of the lower extremity after weaning from ECMO, and no pulmonary embolism was found in the chest X-ray. The success rate of weaning from ECMO was 69.23%(18/26). The D-dimer decreased [584(348,2 107)µg/L vs 1 440(631,2 916)µg/L, P=0.014] and the left ventricular ejection fraction (LVEF) increased (51.4%±8.5% vs 46.9%±10.6%, P=0.013) on the next day after weaning. There were significant differences in heart rate (HR), central venous pressure (CVP), oxygenation index and lactate (Lac) during the PCRTO in the group which involved the cases of the 8 failed experiments (all P<0.05). Compared with the failure group, there were significant differences in age, blood flow rate, CVP before the test, HR, pulse oxygen saturation(SpO(2)), CVP, Lac and oxygenation index after the test, and the variations of SpO(2), CVP and Lac. Conclusion: PCRTO is a simple, reversible, safe and effective weaning method. It can be used in the process of VA-ECMO withdrawal in adult patients.
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Procedimientos Quirúrgicos Cardíacos , Oxigenación por Membrana Extracorpórea , Anciano , Femenino , Hemodinámica , Humanos , Masculino , Persona de Mediana Edad , Volumen Sistólico , Función Ventricular IzquierdaRESUMEN
To assess the usefulness and applications of machine vision (MV) and machine learning (ML) techniques that have been used to develop a single cell-based phenotypic (live and fixed biomarkers) platform that correlates with tumor biological aggressiveness and risk stratification, 100 fresh prostate samples were acquired, and areas of prostate cancer were determined by post-surgery pathology reports logged by an independent pathologist. The prostate samples were dissociated into single-cell suspensions in the presence of an extracellular matrix formulation. These samples were analyzed via live-cell microscopy. Dynamic and fixed phenotypic biomarkers per cell were quantified using objective MV software and ML algorithms. The predictive nature of the ML algorithms was developed in two stages. First, random forest (RF) algorithms were developed using 70% of the samples. The developed algorithms were then tested for their predictive performance using the blinded test dataset that contained 30% of the samples in the second stage. Based on the ROC (receiver operating characteristic) curve analysis, thresholds were set to maximize both sensitivity and specificity. We determined the sensitivity and specificity of the assay by comparing the algorithm-generated predictions with adverse pathologic features in the radical prostatectomy (RP) specimens. Using MV and ML algorithms, the biomarkers predictive of adverse pathology at RP were ranked and a prostate cancer patient risk stratification test was developed that distinguishes patients based on surgical adverse pathology features. The ability to identify and track large numbers of individual cells over the length of the microscopy experimental monitoring cycles, in an automated way, created a large biomarker dataset of primary biomarkers. This biomarker dataset was then interrogated with ML algorithms used to correlate with post-surgical adverse pathology findings. Algorithms were generated that predicted adverse pathology with >0.85 sensitivity and specificity and an AUC (area under the curve) of >0.85. Phenotypic biomarkers provide cellular and molecular details that are informative for predicting post-surgical adverse pathologies when considering tumor biopsy samples. Artificial intelligence ML-based approaches for cancer risk stratification are emerging as important and powerful tools to compliment current measures of risk stratification. These techniques have capabilities to address tumor heterogeneity and the molecular complexity of prostate cancer. Specifically, the phenotypic test is a novel example of leveraging biomarkers and advances in MV and ML for developing a powerful prognostic and risk-stratification tool for prostate cancer patients.
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BACKGROUND: The microchannel heat exchange system has several advantages and can be used to enhance heat transfer for vitrification. OBJECTIVE: To evaluate the microchannel cooling method and to analyze the effects of key parameters such as channel structure, flow rate and sample size. MATERIALS AND METHODS: A computational flow dynamics model is applied to study the two-phase flow in microchannels and its related heat transfer process. The fluid-solid coupling problem is solved with a whole field solution method (i.e., flow profile in channels and temperature distribution in the system being simulated simultaneously). RESULTS AND CONCLUSION: Simulation indicates that a cooling rate >104 C/min is easily achievable using the microchannel method with the high flow rate for a board range of sample sizes. Channel size and material used have significant impact on cooling performance. Computational flow dynamics is useful for optimizing the design and operation of the microchannel system.
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Células/metabolismo , Frío , Criopreservación/métodos , Modelos Teóricos , Especificidad de Órganos , Vitrificación , Calor , Transición de Fase , Factores de TiempoRESUMEN
Objective: To assess the factors associated with outcome of patients undergoing extracorporeal membrane oxygenation (ECMO) in a large ECMO center. Methods: Patients aged >18 years who received ECMO support for postcardiotomy cardiogenic shock were identified between January 2011 and December 2015. One hundred and seventy-seven patients (64.8%) successfully weaned from ECMO. These patients were divided into two groups depending on whether they could survive to hospital discharge: the survival group (group S, n=119) and death group (group D, n=58). Multivariate logistic regression was performed to identify risk factors independently associated with in-hospital mortality. Results: Compared to those from group D, patients in group S exhibited a younger age[(53.4±11.7) vs (58.9±11.5) years], a lower inotrope score at the beginning of ECMO [25(15, 60) vs 35.0(23, 60)], a lower average platelets transfusion [4.0(2.0, 5.2) vs 5.0(3.0, 7.2)U] (all P<0.05). There were shorter duration of ECMO support [95.0(73.0, 131.0) vs 120.0(95.8, 160.2) h], shorter ventilation time [137.0(70.0, 236.8) vs 215.0(164.0, 305.0) h], shorter stay in ICU [182.0(140.0, 236.0) vs 259.0(207.0, 382.0) h] and longer hospital stay after weaned from ECMO [14(11, 24) vs 8(4, 16) d] in group S patients compared to those in group D (all P<0.05). Age>65 years (P=0.046), neurologic complications (P<0.001) and lower extremity ischemia (P<0.001) during ECMO support, left ventricular ejection fraction<35% (P=0.011) and central venous pressure (CVP)>12 cmH(2)O(P=0.018) when weaned from ECMO, and the multi-organ function failure (P<0.001) after weaned from ECMO were independently associated with in-hospital mortality. Conclusions: Neurologic complications and lower extremity ischemia that occurred during ECMO, multi-organ function failure after weaned from ECMO had a significant impact on in-hospital mortality. Further studies are needed to prevent neurologic complications and lower extremity ischemia in these patients. Interventions that could reduce these complications may improve outcome.
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Oxigenación por Membrana Extracorpórea , Mortalidad Hospitalaria , Choque Cardiogénico/mortalidad , Adulto , Anciano , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Choque Cardiogénico/terapia , Resultado del TratamientoRESUMEN
UNLABELLED: About 1051 endophytic fungi were isolated from leaves, branches, barks and stems of Cephalotaxus hainanensis Li from four sites in Hainan, China. The fungi were identified as 21 genera by morphology and ITS sequences. One dominant species was Phomopsis quercella in Hainan Tropical Botanical Garden and Bawangling Nature Reserve, with relative frequency of 42·06 and 34·88% respectively. Another dominant species was Colletotrichum boninense in Wuzhishan and Jianfengling Nature Reserves, with relative frequency of 36·84 and 46·97% respectively. Among the selected 21 endophytic fungi, 17 strains (80·95%) had activity against at least one pathogenic bacteria, and 14 strains (66·67%) exhibited activity against at least one fungal pathogens. Neonectria macroconidialis showed strong inhibition against Staphylococcus aureus (inhibition zone being 20 mm), Bacillus subtilis (14 mm) and Streptococcus agalactiae (28 mm). Xylaria sp. showed strong inhibition against Escherichia coli (20 mm), Rhizoctonia solani (20 mm) and Sclerotinia sclerotiorum (17 mm). Verticillium bulbillosum showed great activity against Strep. agalactiae (32 mm) and Fusarium oxysporum (22 mm). These endophytic fungi showed potentials in medicine development. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi from medicinal plants are an important source of novel and viable drugs. Cephalotaxus hainanensis Li is well known for leukaemia treatment and its endophytic fungi were isolated to investigate the diversity and antimicrobial activity. It was found that Ce. hainanensis Li had rich endophytic fungi, and some fungi showed strong antimicrobial activity against certain pathogens. These fungi can be used in medicine development.
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Antiinfecciosos/metabolismo , Cephalotaxus/microbiología , Endófitos/metabolismo , Plantas Medicinales/microbiología , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Ascomicetos/clasificación , Ascomicetos/efectos de los fármacos , Bacillus subtilis/efectos de los fármacos , China , Colletotrichum/efectos de los fármacos , Endófitos/aislamiento & purificación , Fusarium/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Hojas de la Planta/microbiología , Tallos de la Planta/microbiología , Staphylococcus aureus/efectos de los fármacosRESUMEN
Adenylyl cyclase 2 (ADCY2), a class B member of adenylyl cyclases, is important in accelerating phosphor-acidification as well as glycogen synthesis and breakdown. Given its distinct role in flesh tenderization after butchering, we cloned and sequenced the ADCY2 gene from Yanbian cattle and assessed its expression in bovine tissues. A 2947 bp nucleotide sequence representing the full-length cDNA of bovine ADCY2 gene was obtained by 5' and 3' remote analysis computations for gene expression. Analyses of the putative protein sequence showed that ADCY2 had high homology among species, except with the non-mammal Oreochromis niloticus. Gene structural domain analyses in humans and rats indicated that the ADCY2 protein had no flaw; only the transmembrane domain was reduced and the CYCc structure domain was shortened. Assessment of ADCY2 expression in bovine tissues by real-time PCR showed that the highest expression was in the testes, followed by the longissimus dorsi, tensor fasciae latae, and latissimus dorsi. These data will serve as a foundation for further insight into the cattle ADCY2 gene.
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Adenilil Ciclasas/biosíntesis , Adenilil Ciclasas/genética , Clonación Molecular , Adenilil Ciclasas/química , Secuencia de Aminoácidos , Animales , Bovinos , ADN Complementario/genética , Expresión Génica , Glucógeno/biosíntesis , Humanos , Ratas , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Bayesian theorem is applied to infer DNA molecular marker genotype (DNA chain type) from its phenotype (electrophoresis band type). The results indicate that large difference often presents in the genotype probability of a molecular marker with incomplete genetic information when it is obtained from the assumption of independence among markers as compared with that inferred from the genotypes of the flanking markers with the complete genetic information and the recombination fractions among them based on the Bayesian theorem. Therefore, before utilizing the marker information, such as in mapping quantitative trait loci (QTL), marker assisted selection (MAS) etc., Bayes' probability of the genotype for all markers with incomplete genetic information must be calculated over the whole genome for every individual. This study provided detailed procedure for the calculation of the Bayes' probability of the unknown DNA genotype. Several extensions were also discussed for the application of the Bayesian theorem in genetics.
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Teorema de Bayes , Marcadores Genéticos , GenotipoRESUMEN
We have examined the interaction of recombinant lily pollen ADF, LlADF1, with actin and found that whilst it bound both G- and F-actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil--a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F-actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF : actin array is replaced by actin : ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co-localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.
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Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Polen/metabolismo , Factores Despolimerizantes de la Actina , Destrina , Magnoliopsida/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin alpha, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin alpha homologue from rice which is in addition to our previously isolated rice importin alpha1a and alpha2, and we have named it rice importin alpha1b. In vitro binding and nuclear import assays using recombinant importin alpha1b protein demonstrate that rice importin alpha1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin alpha genes revealed that the genes were not only differentially expressed but that they also responded to dark-adaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin alpha1b. These data suggest that each of the different rice importin alpha proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin alpha contribute to the regulation of plant nuclear protein transport.
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Proteínas de Arabidopsis , Proteínas Portadoras/metabolismo , Señales de Localización Nuclear , Proteínas Nucleares/genética , Oryza/genética , Proteínas de Plantas/metabolismo , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Núcleo Celular/metabolismo , Clonación Molecular , ADN Complementario , Digitonina/farmacología , Células HeLa , Humanos , Carioferinas , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteínas de Plantas/química , Unión Proteica , Transporte de Proteínas , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
In plants there are three microtubule arrays involved in cellular morphogenesis that have no equivalent in animal cells. In animals, microtubules are decorated by another class of proteins - the structural MAPS - which serve to stabilize microtubules and assist in their organization. The best-studied members of this class in plants are the MAP-65 proteins that can be purified together with plant microtubules after several cycles of polymerization and depolymerization. Here we identify three similar MAP-65 complementary DNAs representing a small gene family named NtMAP65-1, which encode a new set of proteins, collectively called NtMAP65-1. We show that NtMAP65-1 protein localizes to areas of overlapping microtubules, indicating that it may function in the behaviour of antiparallel microtubules in the mitotic spindle and the cytokinetic phragmoplast.
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Nicotiana/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Secuencia de Aminoácidos , Compartimento Celular , ADN Complementario/genética , ADN de Plantas/genética , Técnica del Anticuerpo Fluorescente , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas de Plantas/aislamiento & purificación , Análisis de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We recently isolated two cDNAs encoding importin 3 homologues (rice importin beta1 and beta2), the first such homologues identified in plants. To address the function of rice importin beta1 in the process of nuclear import of proteins, we carried out in vitro binding and nuclear import assays. Recombinant protein of rice importin beta1 assembled a complex (PTAC) with rice importin alpha1 and NLS protein, and also bound to the nuclear envelope of tobacco BY-2 cells. Ran-GTP, but not Ran-GDP, interacted with rice importin beta1 and dissociated the heterodimer formed between rice importin alpha1 and rice importin beta1. An in vitro nuclear import assay using digitonin-permeabilized HeLa cells revealed that rice importin beta1 can mediate nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data strongly suggest that rice importin beta1 functions as a component of the NLS receptor in plant cells.
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Proteínas Nucleares/metabolismo , Oryza/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , ADN Complementario , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Carioferinas , Plantas Tóxicas , Unión Proteica , Proteínas Recombinantes/metabolismo , Nicotiana , Proteína de Unión al GTP ranRESUMEN
The nuclear transport of proteins is mediated by the complex of importin-alpha and importin-beta. We isolated two cDNAs encoding importin-beta from rice. A rice importin-beta was demonstrated to interact with rice GST-importin-alpha fusion proteins. The presence of two importin-beta genes was shown for the first time among a variety of eukaryotes.
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Genes de Plantas , Proteínas Nucleares/genética , Oryza/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Plantas , Humanos , Carioferinas , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Nuclear import of most nuclear proteins is initiated by recognition of the nuclear localization signal (NLS) by importin alpha. We recently isolated an importin alpha homologue from rice (rice importin alpha1) and demonstrated that transcription of the gene is down-regulated by light in rice leaves. To address the function of rice importin alpha1 in the process of nuclear import of proteins, we performed in vitro binding and nuclear import assays. The rice importin alpha1 showed specific binding to fusion proteins containing either monopartite or bipartite NLSs, but not to a fusion protein containing a Matalpha-2-type NLS, suggesting that there exists selective binding of rice importin alpha1 to different plant NLSs. The rice importin alpha1 is also capable of forming a complex with mouse importin beta and NLS protein in vitro. An in vitro nuclear import assay using permeabilized HeLa cells revealed that rice importin alpha1, in conjunction with other vertebrate transport factors, mediates the nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data provide strong, direct evidence suggesting that rice importin alpha1 functions as a component of the NLS receptor in plant cells.
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Proteínas Nucleares/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Clonación Molecular , Cartilla de ADN , Escherichia coli , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Carioferinas , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Datos de Secuencia Molecular , Señales de Localización Nuclear , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Oryza/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Maize actin-depolymerizing factor, ZmADF, binds both G- and F-actin and enhances in vitro actin dynamics. Evidence from studies on vertebrate ADF/cofilin supports the view that this class of protein responds to intracellular and extracellular signals and causes actin reorganization. As a test to determine whether such signal-responsive pathways existed in plants, this study addressed the ability of maize ADF to be phosphorylated and the likely effects of such phosphorylation on its capacity to modulate actin dynamics. It is shown that maize ADF3 (ZmADF3) can be phosphorylated by a calcium-stimulated protein kinase present in a 40-70% ammonium sulphate fraction of a plant cell extract. Phosphorylation is shown to be on Ser6, which is only one of nine amino acids that are fully conserved among the ADF/cofilin proteins across distantly related species. In addition, an analogue of phosphorylated ZmADF3 created by mutating Ser6 to Asp6 (zmadf3-4) does not bind G- or F-actin and has little effect on the enhancement of actin dynamics. These results are discussed in context of the previously observed actin reorganization in root hair cells.
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Actinas/metabolismo , Calcio/fisiología , Proteínas de Microfilamentos/metabolismo , Proteínas Quinasas/metabolismo , Serina/metabolismo , Zea mays/metabolismo , Factores Despolimerizantes de la Actina , Secuencia de Aminoácidos , Destrina , Gelsolina/metabolismo , Cinética , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Extractos Vegetales/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3-1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3-2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long alpha-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 A, respectively, with corresponding bond angles of 99.5 degrees and 113 degrees, close to the optimum for a strong hydrogen bond.
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Actinas/metabolismo , Proteínas Portadoras/genética , Proteínas del Citoesqueleto , Proteínas de Plantas/metabolismo , Zea mays/metabolismo , Factores Despolimerizantes de la Actina , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Secuencia Conservada , Destrina , Dimerización , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Conformación Proteica , Alineación de Secuencia , Tirosina/genética , Zea mays/genéticaRESUMEN
The maize actin depolymerizing factor, ZmADF3, binds G- and F-actin, and increases in vitro actin dynamics. Polyclonal antibodies have been raised against ZmADF3 and these detect a single band of approximately 17 kDa in all maize tissues examined, with the exception of pollen. In the development of root hairs, the distribution of ZmADF3 is related to actin reorganization. In the early stages of hair development, ZmADF3 is distributed throughout the cytoplasm. As the hair emerges and the microfilament bundles redirect to the outgrowth there is a simultaneous redistribution of ZmADF3 which now concentrates at the tip of the emerging hair and remains in this position as elongation proceeds. These observations show that ZmADF3 localizes to a region where actin is being remodelled during tip growth. After cytochalasin D treatment which disrupts actin filaments, short rods of ZmADF3 and actin appear in the nucleus suggesting that ZmADF3 may function by guiding actin to sites of actin polymerization.
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Núcleo Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Zea mays/fisiología , Factores Despolimerizantes de la Actina , Actinas/efectos de los fármacos , Actinas/metabolismo , Citocalasina D/farmacología , Citoplasma/metabolismo , Destrina , Cinética , Raíces de Plantas/fisiología , Proteínas Recombinantes/metabolismoRESUMEN
In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.