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We present a re-examination of mass spectral data obtained from the Pioneer Venus Large Probe Neutral Mass Spectrometer. Our interpretations of differing trace chemical species are suggestive of redox disequilibria in Venus' middle clouds. Assignments to the data (at 51.3 km) include phosphine, hydrogen sulfide, nitrous acid, nitric acid, carbon monoxide, hydrochloric acid, hydrogen cyanide, ethane, and potentially ammonia, chlorous acid, and several tentative PxOy species. All parent ions were predicated upon assignment of corresponding fragmentation products, isotopologues, and atomic species. The data reveal parent ions at varying oxidation states, implying the presence of reducing power in the clouds, and illuminating the potential for chemistries yet to be discovered. When considering the hypothetical habitability of Venus' clouds, the assignments reveal a potential signature of anaerobic phosphorus metabolism (phosphine), an electron donor for anoxygenic photosynthesis (nitrite), and major constituents of the nitrogen cycle (nitrate, nitrite, ammonia, and N2).
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BACKGROUND: Lung transplant recipients are at high risk of skin cancer, but precise annual incidence rates of treated skin cancers per patient are unknown. OBJECTIVES: To perform a prospective assessment of the total burden of histologically confirmed squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) and associated factors in lung transplant recipients. METHODS: A population-based cohort of 125 Queensland lung transplant recipients aged 18 years and over, recruited between 2013 and 2015, were followed to the end of 2016. All underwent dermatological skin examinations at baseline and annually thereafter and patients self-reported all interim treated skin cancers, which were verified against pathology databases. Standard skin cancer risk factors were obtained via questionnaire, and details of medications were acquired from hospital records. RESULTS: During a median follow-up time of 1·7 years, 29 (23%) and 30 (24%) lung transplant recipients with a median duration of immunosuppression of 3·3 years developed SCC and BCC, respectively. The general population age-standardized incidence rates of SCC and BCC were 201 and 171 per 1000 person-years, respectively (based on first primary SCC or BCC during follow-up); however, on accounting for multiple primary tumours, corresponding incidence rates were 447 and 281 per 1000 person-years. Risk of multiple SCCs increased around sixfold in those aged ≥ 60 years and in those with previous skin cancer, and increased around threefold in those treated with the antifungal medication voriconazole. Multiple BCC risk rose threefold from age 60 years and tenfold for patients with previous skin cancer. CONCLUSIONS: Lung transplant recipients have very high incidence of multiple primary skin cancers. Close surveillance and assiduous prevention measures are essential. Linked Comment: Proby and Harwood. Br J Dermatol 2020; 183:416-417.
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Carcinoma Basocelular , Neoplasias Cutáneas , Adolescente , Adulto , Anciano , Carcinoma Basocelular/epidemiología , Humanos , Incidencia , Pulmón , Persona de Mediana Edad , Estudios Prospectivos , Queensland/epidemiología , Factores de Riesgo , Neoplasias Cutáneas/epidemiología , Receptores de TrasplantesRESUMEN
OBJECTIVE: The aim of this study was to establish rotational thromboelastometry (ROTEM® ) baseline parameters in labouring women at term gestation. The secondary aim was to compare these reference ranges with those from previous studies on labouring women and from the manufacturer. DESIGN: A prospective, observational study. SETTING: Tertiary referral hospital. PARTICIPANTS: Healthy women in labour. METHODS: Ethics approval was granted for an opt-out recruitment approach. ROTEM® testing was performed in labouring women at term gestation. Women with any condition affecting coagulation were excluded. ROTEM® Delta reference ranges were derived by calculating the 2.5% and 97.5% centiles for INTEM/EXTEM/FIBTEM parameters including amplitude at 5 minutes (A5), coagulation time (CT) and maximum clot firmness (MCF). MAIN OUTCOME MEASURES: ROTEM® parameters were measured in labouring women before delivery. The following tests were performed: FIBTEM, EXTEM and INTEM. RESULTS: One hundred and twenty-one women met the inclusion criteria, with a mean (± SD) age of 29.6 ± 5.4 years and median (interquartile range) gestation of 39.4 weeks (37.4-40.4 weeks). Seventy-five (62.0%) women were nulliparous and 71 (58.7%) delivered vaginally. The median and interquartile ranges for selected ROTEM® parameters were: FIBTEM A5, 21 mm (IQR 18-23 mm); EXTEM A5, 55 mm (52-58 mm); and EXTEM CT, 52 seconds (48-56 seconds). CONCLUSIONS: The FIBTEM/EXTEM/INTEM amplitudes were higher than the manufacturer's reference ranges for non-obstetric patients. The FIBTEM MCF upper and lower limits were higher and the EXTEM/INTEM CT was shorter and narrower in range. This study provides reference ranges for ROTEM® values in healthy labouring women at term gestation with uncomplicated pregnancies. TWEETABLE ABSTRACT: This is the first study to report on ROTEM® reference ranges with over 120 healthy labouring women of normal weight at term gestation.
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Trabajo de Parto/fisiología , Diagnóstico Prenatal/estadística & datos numéricos , Tromboelastografía/estadística & datos numéricos , Adulto , Femenino , Voluntarios Sanos , Humanos , Embarazo , Diagnóstico Prenatal/métodos , Estudios Prospectivos , Valores de Referencia , Tromboelastografía/métodosRESUMEN
Present-day Venus is an inhospitable place with surface temperatures approaching 750K and an atmosphere 90 times as thick as Earth's. Billions of years ago the picture may have been very different. We have created a suite of 3-D climate simulations using topographic data from the Magellan mission, solar spectral irradiance estimates for 2.9 and 0.715 Gya, present-day Venus orbital parameters, an ocean volume consistent with current theory, and an atmospheric composition estimated for early Venus. Using these parameters we find that such a world could have had moderate temperatures if Venus had a rotation period slower than ~16 Earth days, despite an incident solar flux 46-70% higher than Earth receives. At its current rotation period, Venus's climate could have remained habitable until at least 715 million years ago. These results demonstrate the role rotation and topography play in understanding the climatic history of Venus-like exoplanets discovered in the present epoch.
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The Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Crambidae) is an invasive stem borer of sugarcane, Saccharum spp., and sorghum, Sorghum bicolor (L.), and poses a threat against the production of dedicated bioenergy feedstocks in the U.S. Gulf Coast region. A 2-yr field study was conducted in Jefferson County, TX, to evaluate yield losses associated with E. loftini feeding on bioenergy and conventional cultivars of sugarcane and sorghum under natural and artificially established E. loftini infestations. Bioenergy sugarcane (energycane) 'L 79-1002' and 'Ho 02-113' and sweet sorghum 'M81E' exhibited reduced E. loftini injury; however, these cultivars, along with high-biomass sorghum cultivar 'ES 5140', sustained greater losses in fresh stalk weight. Negative impacts to sucrose concentration from E. loftini injury were greatest in energycane, high-biomass sorghum, and sweet sorghum cultivars. Even under heavy E. loftini infestations, L 79-1002, Ho 02-113, and 'ES 5200' were estimated to produce more ethanol than all other cultivars under suppressed infestations. ES 5200, Ho 02-113, and L 79-1002 hold the greatest potential as dedicated bioenergy crops for production of ethanol in the Gulf Coast region; however, E. loftini management practices will need to be continued to mitigate yield losses.
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Herbivoria , Mariposas Nocturnas/fisiología , Saccharum/crecimiento & desarrollo , Sorghum/crecimiento & desarrollo , Animales , Biomasa , Conducta Alimentaria , Larva/crecimiento & desarrollo , Larva/fisiología , Mariposas Nocturnas/crecimiento & desarrollo , Densidad de Población , TexasRESUMEN
We present the first published method to convert data obtained by the Pioneer Venus Large Probe Neutral Mass Spectrometer (LNMS) into units of mixing ratio (ppm) and volume percent (v%) against CO2 and N2, the dominant Venus atmospheric gases, including conversion to density (kg m-3). These unit conversions are key to unlocking the untapped potential of the data, which represents a significant challenge given the scant calibration data in the literature. Herein, we show that our data treatments and conversions yield mixing ratios and volume percent values for H2O, N2, and SO2 that are within error to those reported for the gas chromatograph (LGC) on the Pioneer Venus Large Probe (PVLP). For the noble gases, we developed strategies to correct for instrument biases by treating the data as a relative scale and using PVLP and Venera-based measurements as calibration points. Together, these methods, conversions, calibrations, and comparisons afford novel unit conversions for the LNMS data and yield unified measures for Venus' atmosphere from the LNMS and LGC on the PVLP.â¢Conversion into mixing ratio (ppm), volume percent (v%), and density (kg m-3).â¢Mixing ratios are expressed against CO2 and N2.â¢LNMS and LGC measurements on the PVLP are consistent.
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Here we examine how our knowledge of present day Venus can inform terrestrial exoplanetary science and how exoplanetary science can inform our study of Venus. In a superficial way the contrasts in knowledge appear stark. We have been looking at Venus for millennia and studying it via telescopic observations for centuries. Spacecraft observations began with Mariner 2 in 1962 when we confirmed that Venus was a hothouse planet, rather than the tropical paradise science fiction pictured. As long as our level of exploration and understanding of Venus remains far below that of Mars, major questions will endure. On the other hand, exoplanetary science has grown leaps and bounds since the discovery of Pegasus 51b in 1995, not too long after the golden years of Venus spacecraft missions came to an end with the Magellan Mission in 1994. Multi-million to billion dollar/euro exoplanet focused spacecraft missions such as JWST, and its successors will be flown in the coming decades. At the same time, excitement about Venus exploration is blooming again with a number of confirmed and proposed missions in the coming decades from India, Russia, Japan, the European Space Agency (ESA) and the National Aeronautics and Space Administration (NASA). Here we review what is known and what we may discover tomorrow in complementary studies of Venus and its exoplanetary cousins.
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The bacterial pathogens Listeria monocytogenes and Shigella flexneri recruit host factors that enable them to use actin polymerization as the driving force to facilitate their spread into neighbouring cells. It is now becoming clear that other pathogens, including viruses, have developed a number of different strategies to use the actin cytoskeleton of the host to their advantage during the infection process.
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Actinas/fisiología , Listeria/química , Listeria/patogenicidad , Shigella flexneri/química , Shigella flexneri/patogenicidad , Virulencia/fisiologíaRESUMEN
In the past decade, studies into the way in which intracellular bacterial pathogens hijack and subvert their hosts have provided many important insights into regulation of the actin cytoskeleton and cell motility, in addition to increasing our understanding of the infection process. Viral pathogens, however, may ultimately unlock more cellular secrets as they are even more dependent on their hosts during their life cycle.
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Citoesqueleto/virología , Fenómenos Fisiológicos de los Virus , Virus/patogenicidad , Animales , Transporte Biológico Activo , Células HeLa , Humanos , Virosis/virologíaRESUMEN
Actin and microtubules represent complex polymer systems that play essential roles during many cellular processes including chromosome segregation, cytokinesis and motility. The dynamic nature of actin and microtubules together with their regulation by a myriad of proteins makes their study both fascinating and challenging. Over the past few years there has been an increasing move towards development of in vitro systems to facilitate the elucidation of the molecular basis of actin and microtubule dependent cell processes. This review focuses on some of the recent developments using in vitro assays to dissect the cellular role of the actin and microtubule cytoskeleton.
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Actinas/fisiología , Microtúbulos/fisiología , Animales , Movimiento Celular , Dineínas/metabolismo , Técnicas In Vitro , Cinesinas/metabolismo , Melanosomas/fisiología , Huso Acromático/fisiologíaRESUMEN
Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signalling cascades to actin polymerization. Here we show that the amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP is responsible for its recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella WIP is recruited by N-WASP. Our observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signalling pathways to recruit the N-WASP-WIP complex. We propose that the N-WASP-WIP complex has a pivotal function in integrating signalling cascades that lead to actin polymerization.
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Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Citoesqueleto de Actina/química , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Biopolímeros/química , Biopolímeros/metabolismo , Proteínas Portadoras/genética , Proteínas del Citoesqueleto , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Movimiento , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Shigella/fisiología , Virus Vaccinia/fisiología , Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Dominios Homologos srcRESUMEN
Vaccinia virus, a close relative of the causative agent of smallpox, exploits actin polymerization to enhance its cell-to-cell spread. We show that actin-based motility of vaccinia is initiated only at the plasma membrane and remains associated with it. There must therefore be another form of cytoplasmic viral transport, from the cell centre, where the virus replicates, to the periphery. Video analysis reveals that GFP-labelled intracellular enveloped virus particles (IEVs) move from their perinuclear site of assembly to the plasma membrane on microtubules. We show that the viral membrane protein A36R, which is essential for actin-based motility of vaccinia, is also involved in microtubule-mediated movement of IEVs. We further show that conventional kinesin is recruited to IEVs via the light chain TPR repeats and is required for microtubule-based motility of the virus. Vaccinia thus sequentially exploits the microtubule and actin cytoskeletons to enhance its cell-to-cell spread.
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Actinas/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Virus Vaccinia/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Estructurales Virales/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Transporte Biológico , Núcleo Celular/metabolismo , Pollos , Ratones , Datos de Secuencia MolecularRESUMEN
Since outbreaks were first detected in grain sorghum, Sorghum bicolor (L.) Moench (Cyperales: Poaceae), in 2013, sugarcane aphid, Melanaphis sacchari Zehntner has become a major annual pest in grain sorghum-producing regions of North America. Economic thresholds have been recommended for susceptible hybrids, but these recommendations may not be suitable for grain sorghum hybrids partially resistant to sugarcane aphid. The objectives were to evaluate the grain yield-aphid density relationship and field-based population growth rates of the aphid on sorghum hybrids susceptible and partially resistant to sugarcane aphid across multiple years, locations, and hybrids. These data verified previously established economic injury levels for susceptible hybrids. The observed maximum aphid density ranged from 6 to 451 aphids per leaf for resistant hybrids and from 67 to 1,025 for susceptible hybrids. Across 50 location-year combinations, the maximum aphid density observed on resistant hybrids decreased by 0-99%, compared to a susceptible hybrid at the same location (mean reduction = 80%). Doubling time for sugarcane aphid populations on partially resistant hybrids was up to 6.4-fold higher than on known susceptible hybrids. For 48 of the 50 location-years, yield loss attributable to sugarcane aphid was not detected on the partially resistant hybrids; therefore, an economic injury level was unable to be estimated. If an economic injury level exists for resistant hybrids, it is likely at an aphid population level that exceeds the levels experienced in this study. It remains prudent to monitor resistant hybrids for unusual leaf decay associated with aphid densities that exceed current economic injury levels used for susceptible hybrids.
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Áfidos , Sorghum , Animales , Grano Comestible , América del Norte , Sorghum/genéticaRESUMEN
Rice (Oryza sativa L.) plants in experimental plots in Beaumont, TX developed symptoms on leaf blades similar to white leaf streak caused by Mycovellosiela oryzae (Deighton & Shaw) Deighton (synonym Ramularia oryzae Deighton & Shaw) (1,3,4) during the late summer of 2009. Symptoms were observed on several rice cultivars and breeding lines including Cocodrie, Cypress, and Lemont. Lesions usually appeared on lower leaves and were approximately 2 to 7 mm long and linear with whitish or grayish centers surrounded by a narrow brown-or-dark brown margin. Symptoms were similar to narrow brown leaf spot caused by Cercospora janseana (Racib.) O. Const. (synonym C. oryzae Miyake) (3,4), but the centers of lesions were slightly wider (up to 2 mm). Symptoms were visible on the upper and lower leaf surfaces. Some lesions on heavily infected leaves were long (10 to 15 × 0.5 to 2 mm) whitish streaks parallel to the midrib. Leaves with typical symptoms were collected and incubated in a petri dish lined with moistened filter paper for 3 to 4 days at room temperature under a 12-h fluorescent photoperiod. Conidiophores were produced on external mycelium growing out through stomata on the lesion surface. Conidiophores were hyaline, straight, and 7 to 22 µm long and 2 to 3 µm wide with conidial scars. Conidia were washed from the lesions, diluted in sterilized distilled water, and placed on acidified potato dextrose agar. After 6 to 7 days of incubation at room temperature, slow-growing colonies were transferred onto potato dextrose agar (PDA). Three isolates were obtained from single-spore cultures. The colonies of these isolates grew similarly and very slowly on PDA and their radial growth averaged 0.8 mm/day at room temperature. The colonies were dense, grayish, and did not produce any pigments. Conidia were formed singly or in chains and measured 12 to 30 µm long. They were hyaline, straight, cylindrical, typically with no or one septum, a few with two to three septa, and had a hilum and tapered ends. Pathogenicity of these three isolates was assessed in greenhouse tests by spraying a conidial suspension (105 conidia/ml) onto 12 plants of each of the cvs. Cocodrie, Cypress and Lemont at the late tillering stage. Inoculum was obtained by harvesting conidia from the colonies grown on PDA for 3 weeks at room temperature under a 12-h fluorescent photoperiod. Plants sprayed with sterilized distilled water served as the controls. All plants were maintained in a humid chamber for 2 days and then grown in a greenhouse at 20 to 31°C. After 15 days, early lesions appeared on inoculated leaf blades; after 4 weeks, typical symptoms similar to those observed in the field developed. Control plants did not exhibit any symptoms. M. oryzae was reisolated from symptomatic plants, confirming that the disease was caused by this pathogen. To our knowledge, this is the first report of white leaf streak on rice in Texas and the second report after Louisiana (2). This disease has been reported in Papua New Guinea, the Solomon Islands, North Borneo, Sierra Leone, and Nigeria (3,4). References: (1) F. C. Deighton and D. Shaw. Trans. Br. Mycol. Soc. 43:515, 1960. (2) A. K. M. Shahjahan et al. Plant Dis. 82:1282, 1998. (3) B. C. Sutton and A. K. M. Shahjahan. Nova Hedwigia 25:197, 1981. (4) R. K. Webster and P. S. Gunnell. Compendium of Rice Diseases. The American Phytopathological Society, St. Paul, MN, 1992.
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BACKGROUND: Rotational thromboelastometry (ROTEM®) is a point-of-care coagulation test. Reference ranges in non-labouring women have recently been established from a cohort of women presenting for elective caesarean delivery using the recommended minimum sample size of 120. This study aimed to present baseline parameters for labouring and non-labouring women and to compare the mean values of these ROTEM® parameters. METHODS: Ethical approval was granted for an opt-out recruitment approach for labouring women and written consent was obtained from non-labouring women (data published previously). ROTEM® testing was performed in these two cohorts at term gestation. Women with any condition affecting coagulation were excluded. ROTEM® Delta reference ranges were derived by calculating the 2.5 and 97.5 percentiles for INTEM/EXTEM/FIBTEM amplitude at 5 min (A5), coagulation time (CT), maximum clot firmness (MCF) and clot formation time (CFT). RESULTS: One hundred and twenty-one labouring and 132 non-labouring women met inclusion criteria. The mean values for selected ROTEM® parameters for labouring and non-labouring women respectively were: FIBTEM A5, 21.05 and 19.7â¯mm (P=0.008); EXTEM A5, 54.8 and 53.2â¯mm (P=0.025); and EXTEM CT, 52.2 and 53.7â¯s (P=0.049). Significant differences between the groups were observed in measures of clotting onset and clot firmness. CONCLUSIONS: We demonstrated a significant decrease in the mean time-to-clotting onset in labouring women compared with non-labouring women. Mean values for measures of clot firmness were greater in labouring women. In comparison to previously established ROTEM® baseline parameters for non-labouring women, this study provides evidence that there is greater hyper-coagulability in labouring women.
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Trabajo de Parto/sangre , Pruebas en el Punto de Atención , Embarazo/sangre , Tromboelastografía/métodos , Adulto , Femenino , Humanos , Valores de ReferenciaRESUMEN
A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia
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Actinas/fisiología , Proteínas del Citoesqueleto , Listeria/patogenicidad , Shigella/patogenicidad , Virus Vaccinia/patogenicidad , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Actinas/metabolismo , Citoesqueleto/microbiología , Escherichia coli/patogenicidad , Imitación Molecular , Proteínas del Tejido Nervioso/fisiología , Polímeros , Proteínas Tirosina Quinasas/metabolismo , Rickettsia/patogenicidad , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The minimal region required for actin binding in the smallest of the three domains of gelsolin (termed Segment 1 or S1) was previously defined by deletion mutagenesis as residues 37-126. Further analysis of NH2-terminal deletions here redefines the minimal functional core as residues 41-126. Amino acid substitutions within this core further elucidate the nature of the interaction of segment 1 with actin. Of 26 point mutants analyzed, 14 reduced the affinity for actin. The charged residues His 119, Arg 120, Glu 121, and Gln 123 appear to be involved in direct interaction with actin. Substitutions of Leu 108, Leu 112, and Val 117 by polar groups all affect the structural stability of segment 1 and thereby reduce binding affinity. In addition replacement of Glu 126 by aspartic acid modifies the physical properties of segment 1 and weakens binding. We have further shown that changing charged residues within the highly conserved pentapeptide sequence LDDYL (residues 108-112) has no effect on actin binding. This sequence, found in a number of different actin binding proteins, does not therefore constitute part of the interaction site. Similarly, substitution of the two acidic residues by basic ones within the DESG motif of segment 1 (residues 96-99, but also found near the COOH terminus of actin) does not impair binding. These results show the dangers of predicting functional sites on the basis of conserved sequences.
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Actinas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Microfilamentos/metabolismo , Acanthamoeba/genética , Actinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al Calcio/genética , Análisis Mutacional de ADN , Gelsolina , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-DirigidaRESUMEN
The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.
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Actinina/química , Actinas/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Microfilamentos/química , Actinina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Unión al Calcio/metabolismo , Gelsolina , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia MolecularRESUMEN
The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.
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Actinina/química , Actinas/química , Conformación Proteica , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestructura , Actinina/metabolismo , Actinina/ultraestructura , Actinas/metabolismo , Actinas/ultraestructura , Animales , Sitios de Unión , Pollos , Gráficos por Computador , Análisis de Fourier , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Electrónica/métodos , Estructura Molecular , Músculos/químicaRESUMEN
Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.