RESUMEN
Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.
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Streptococcus pneumoniae , Estreptococos Viridans , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Estreptococos Viridans/genética , Bases de Datos de Ácidos Nucleicos , TaiwánRESUMEN
OBJECTIVE: This study was conducted to investigate key long noncoding RNAs (lncRNAs) involved in competitive endogenous RNA (ceRNA) network associated with laryngeal squamous cell carcinoma (LSCC). MATERIALS AND METHODS: Three mRNA datasets, two miRNA datasets, and one lncRNA dataset of LSCC were downloaded from GEO database. Following the identification of differentially expressed mRNAs (DEmRNAs), (microRNAs) miRNAs (DEmiRNAs), and lncRNAs (DElncRNAs) in LSCC compared with adjacent tissues, functional enrichment of DEmRNAs was performed. Then, construction of the ceRNA (DElncRNA-DEmiRNA-DEmRNA) regulatory network and functional analyses of all DEmRNAs in ceRNA regulatory network were conducted. Quantitative real-time polymerase chain reactions (qRT-PCR) were used to detect the expression levels of selected DEmRNAs, DEmiRNAs, and DElncRNAs. RESULTS: A total of 3449 DEmRNAs, 40 DEmiRNAs, and 100 DElncRNAs were identified in LSCC. The ceRNA networks, which contained 132 DElncRNA-DEmiRNA pairs and 287 DEmiRNA-DEmRNA pairs, involving 44 lncRNAs, 3 miRNAs, and 271 mRNAs, were obtained. DEmRNAs in ceRNA regulatory networks were significantly enriched in pathways in cancer, prostate cancer, and aldosterone-regulated sodium reabsorption. Except for HCG22 and hsa-miR-1246, expressions of the others in the qRT-PCR results played the same pattern with that in our integrated analysis, generally. CONCLUSIONS: We concluded that HCG22/EGOT-hsa-miR-1275-FAM107A and HCG22/EGOT-hsa-miR-1246-Glycerol-3-phosphate dehydrogenase 1 like interaction pairs may play a central role in LSCC.
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Neoplasias de Cabeza y Cuello , MicroARNs , ARN Largo no Codificante , Masculino , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , ARN Largo no Codificante/genética , Redes Reguladoras de Genes , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias de Cabeza y Cuello/genética , Regulación Neoplásica de la Expresión Génica/genéticaRESUMEN
The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.
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Complejos de ATP Sintetasa/antagonistas & inhibidores , Antineoplásicos/farmacología , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/metabolismo , ARN Largo no Codificante/metabolismo , Complejos de ATP Sintetasa/genética , Complejos de ATP Sintetasa/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Membrana Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Gefitinib/farmacología , Ontología de Genes , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteómica , ARN Largo no Codificante/genética , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Espectrometría de Masas en Tándem , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.
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Catarata/tratamiento farmacológico , Catarata/metabolismo , Lanosterol/farmacología , Lanosterol/uso terapéutico , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/tratamiento farmacológico , Adulto , Secuencia de Aminoácidos , Amiloide/química , Amiloide/efectos de los fármacos , Amiloide/metabolismo , Amiloide/ultraestructura , Animales , Secuencia de Bases , Catarata/congénito , Catarata/genética , Catarata/patología , Línea Celular , Niño , Cristalinas/química , Cristalinas/genética , Cristalinas/metabolismo , Cristalinas/ultraestructura , Perros , Femenino , Humanos , Lanosterol/administración & dosificación , Cristalino/efectos de los fármacos , Cristalino/metabolismo , Cristalino/patología , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestructura , Linaje , Agregación Patológica de Proteínas/patologíaRESUMEN
Introduction: We studied the impact of vibratory stimulation on the electrophysiological features of digital sensory nerve action potential (SNAP). Methods: The antidromic digit 3 SNAP was recorded in 19 healthy adults before, during, and after applying a vibration to either 3rd or 5th metacarpal phalangeal joint (MCPJ) at 60 Hz and amplitude of 2 mm. 100% supramaximal stimulus intensity was performed in 5 subjects (randomly selected from the 19 subjects) where the SNAP sizes were recorded. Results: The amplitude of digit 3 SNAP declined to 58.9 ± 8.6% when a vibration was applied to MCPJ digit 3. These impacts did not change by increasing the electrical stimulus intensity. The SNAP regained its baseline value immediately after the cessation of vibration stimulation. The magnitude of size reduction of digit 3 SNAP was less when vibration was moved to from MCPJ of digit 3 to MCPJ of digit 5. Discussion. The marked drop of the SNAP size during vibratory stimulation reflects the decreased responsiveness of Aß afferents to electrical stimulation, which deserve further investigation in the study of focal vibration in neurorehabilitation.
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Potenciales de Acción/fisiología , Dedos/inervación , Dedos/fisiología , Nervio Mediano/fisiología , Células Receptoras Sensoriales/fisiología , Vibración , Adulto , Estimulación Eléctrica/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Conducción Nerviosa/fisiología , Proyectos Piloto , Adulto JovenRESUMEN
Cataract, a crystallin aggregation disease, is the leading cause of human blindness worldwide. Surgery is the only established treatment of cataracts and no anti-cataract drugs are available thus far. Recently lanosterol and 25-hydroxycholesterol have been reported to redissolve crystallin aggregates and partially restore lens transparency in animals. However, the efficacies of these two compounds have not been quantitatively studied ex vivo using patient tissues. In this research, we developed a quantitative assay applicable to efficacy validations and mechanistic studies by a protocol to isolate protein aggregates from the surgically removed cataractous human lens. Our results showed that both compounds were effective for human cataractous samples with EC50 values at ten micromolar level. The efficacies of both compounds strongly depended on cataract severity. Lanosterol and 25-hydroxycholesterol were two mechanistically different lead compounds of anti-cataract drug design.
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Catarata/patología , Hidroxicolesteroles/farmacología , Lanosterol/farmacología , Cristalino/metabolismo , Agregación Patológica de Proteínas/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Fluorescencia , Humanos , Cristalino/patología , Masculino , Persona de Mediana EdadRESUMEN
ß/γ-Crystallins are predominant structural proteins in vertebrate lens with unique properties of extremely high solubility, long-term stability and resistance to UV damage. Four conserved Trp residues in ß/γ-crystallins account for UV absorbance and thereafter fluorescence quenching to avoid photodamage. Herein we found that ßB2-crystallin Trp fluorescence was greatly enhanced by the introduction of an extra unquenched Trp fluorophore by cataract-associated mutations S31W and R145W. Both mutations impaired oligomerization, decreased stability and promote thermal aggregation, while S31W was more deleterious. S31W accelerated ßB2-crystallin aggregation under UV damaging conditions, whereas R145W delayed. These observations suggested that the introduction of an extra Trp fluorophore had complicated effects on ßB2-crystallin stability and aggregation against various stresses. Our findings highlight that the number of Trp fluorophores in ß/γ-crystallin is evolutionarily optimized to exquisitely perform their structural roles in the lens.
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Catarata/genética , Triptófano/química , Cadena B de beta-Cristalina/genética , Cadena B de beta-Cristalina/metabolismo , Evolución Molecular , Fluorescencia , Humanos , Simulación de Dinámica Molecular , Desnaturalización Proteica , Estabilidad Proteica , Espectrofotometría Ultravioleta , Rayos Ultravioleta , Cadena B de beta-Cristalina/químicaRESUMEN
In this study, a universal protein expression enhancement RNA tool, termed RNAe, was developed by modifying a recently discovered natural long non-coding RNA. At the moment, RNAe is the only technology for gene expression enhancement, as opposed to silencing, at the post-transcriptional level. With this technology, an expression enhancement of 50-1000% is achievable, with more than 200% enhancement achieved in most cases. This work identified the sufficient and necessary element for RNAe function, which was found to be merely 300 nucleotides long and was named minRNAe. It contains a 72-nt 5' pairing sequence which determines the specificity, a 167-nt short non-pairing interspersed nuclear element (SINE) B2 sequence which enhances ribosome recruitment to the target mRNA, and a poly(A) tail, provided together on a plasmid bearing the appropriate sequences. Cellular delivery of RNAe was achieved using routine transfection. The RNAe platform was validated in several widely-used mammalian cell lines. It was proven to be efficient and flexible in specifically enhancing the expression of various endogenous and exogenous proteins of diverse functions in a dose-dependent manner. Compared to the expression-inhibitory tool RNAi, the RNAe tool has a comparable effect size, with an enhancing as opposed to inhibitory effect. One may predict that this brand new technology for enhancing the production of proteins will find wide applications in both research and biopharmaceutical production.
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Regulación de la Expresión Génica , Biosíntesis de Proteínas , Ingeniería de Proteínas/métodos , ARN Largo no Codificante/química , Formación de Anticuerpos , Línea Celular , Vectores Genéticos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Proteómica , ARN sin Sentido/química , Secuencias Repetitivas de Ácidos Nucleicos , Ribosomas/metabolismoRESUMEN
Vertebrate lens is one of the tissues with the highest soluble protein concentration. The predominant soluble proteins in lens fiber cells are crystallins, and among them, α-crystallins belong to the small heat shock protein family with chaperone-like activity. Although α-crystallins are highly soluble in waters, α-crystallins have been detected in the membrane-bound fraction of lens, which will increase in the aged or cataractous lens. In this research, we found αA-crystallin exhibited a complex thermal transition with remarkable changes in secondary and quaternary structures. Treatment of αA-crystallin at high temperatures induced larger oliogomers with higher hydrophobic exposure. Both heat-treated and untreated αA-crystallin could insert into lipid monolayer directly as revealed by monolayer surface pressure experiments. Heat-treatment facilitated the membrane insertion of αA-crystallin and increased the membrane-bound fraction in the cells. The membrane-binding ability of αA-crystallin could be altered by cataract-causing mutations R116C, R116H and Y118D. Our results suggested that the irreversible changes in oligomer size induced by various stresses might promote the membrane association of αA-crystallin and therefore might play a role in aged cataract. Alternations in the membrane binding ability of α-crystallins might be important to the understanding of both aged and congenital cataracts.
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Membrana Celular/química , Cristalinas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Catarata/metabolismo , Bovinos , Cromatografía , ADN Complementario/metabolismo , Células HeLa , Proteínas de Choque Térmico/química , Humanos , Lípidos/química , Microscopía Fluorescente , Mutación , Fosfatidilserinas/química , Presión , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/química , TemperaturaRESUMEN
AIM: Caveolin-1 (cav-1) is a major multifunctional scaffolding protein of caveolae. Cav-1 is primarily expressed in mesangial cells, renal proximal tubule cells and podocytes in kidneys. Recent evidence shows that the functional connections between cav-1 and ROS play a key role in many diseases. In this study we investigated whether regulating the functional connections between cav-1 and ROS in kidneys contributed to the beneficial effects of curcumin in treating diabetic nephropathy in vitro and in vivo. METHODS: Cultured mouse podocytes (mpc5) were incubated in a high glucose (HG, 30 mmol/L) medium for 24, 48 or 72 h. Male rats were injected with STZ (60 mg/kg, ip) to induce diabetes. ROS generation, SOD activity, MDA content and caspase-3 activity in the cultured cells and kidney cortex homogenate were determined. Apoptotic proteins and cav-1 phosphorylation were analyzed using Western blot analyses. RESULTS: Incubation in HG-containing medium time-dependently increased ROS production, oxidative stress, apoptosis, and cav-1 phosphorylation in podocytes. Pretreatment with curcumin (1, 5, and 10 µmol/L) dose-dependently attenuated these abnormalities in HG-treated podocytes. Furthermore, in HG-containing medium, the podocytes transfected with a recombinant plasmid GFP-cav-1 Y14F (mutation at a cav-1 phosphorylation site) exhibited significantly decreased ROS production and apoptosis compared with the cells transfected with empty vector. In diabetic rats, administration of curcumin (100 or 200 mg/kg body weight per day, ig, for 8 weeks) not only significantly improved the renal function, but also suppressed ROS levels, oxidative stress, apoptosis and cav-1 phosphorylation in the kidneys. CONCLUSION: Curcumin attenuates high glucose-induced podocyte apoptosis in vitro and diabetic nephropathy in vivo partly through regulating the functional connections between cav-1 phosphorylation and ROS.
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Apoptosis/efectos de los fármacos , Caveolina 1/metabolismo , Curcumina/farmacología , Glucosa/metabolismo , Podocitos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones , Estrés Oxidativo , Fosforilación , Podocitos/citología , Podocitos/metabolismo , Ratas WistarRESUMEN
INTRODUCTION: Sensory neuronopathy (SNN) mimics distal sensory axonopathy. The conventional H-reflex elicited by tibial nerve stimulation (tibial H-reflex) is usually abnormal in both conditions. We evaluated the proximally evoked soleus H-reflex in response to S1 nerve root stimulation (S1 foramen H-reflex) in SNN. METHODS: Eleven patients with SNN and 6 with distal sensory axonopathy were studied. Tibial and S1 foramen H-reflexes were performed bilaterally in each patient. RESULTS: Tibial and S1 foramen H-reflexes were absent bilaterally in all patients with SNN. In the patients with distal sensory axonopathy, tibial H-reflexes were absent in 4 and demonstrated prolonged latencies in 2, but S1 foramen H-reflexes were normal. CONCLUSIONS: Characteristic absence of the H-reflex after both proximal and distal stimulation reflects primary loss of dorsal root ganglion (DRG) neurons and the distinct non-length-dependent impairment of sensory nerve fibers in SNN.
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Ganglios Espinales/fisiopatología , Reflejo H/fisiología , Músculo Esquelético/fisiopatología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Trastornos de la Sensación/fisiopatología , Células Receptoras Sensoriales/fisiología , Raíces Nerviosas Espinales/fisiopatología , Adulto , Anciano , Estimulación Eléctrica/instrumentación , Estimulación Eléctrica/métodos , Electromiografía/instrumentación , Electromiografía/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Receptoras Sensoriales/patología , Nervio Tibial/fisiopatología , Adulto JovenRESUMEN
INTRODUCTION: Sensory neuronopathy (SNN) is a distinctive subtype of peripheral neuropathies, specifically targeting dorsal root ganglion (DRG). We utilized MRI to demonstrate the imaging characteristics of DRG, spinal cord (SC), and brachial plexus at C7 level in SNN. METHODS: We attempted multiple-echo data image combination (MEDIC) and turbo inversion recovery magnitude (TIRM) methods in nine patients with sensory neuronopathy and compared with those in 16 disease controls and 20 healthy volunteers. All participants underwent MRI for the measurement of DRG, posterior column (PC), lateral column, and spinal cord area (SCA) at C7 level. DRG diameters were obtained through its largest cross section, standardized by dividing sagittal diameter of mid-C7 vertebral canal. We also made comparisons of standardized anteroposterior diameter (APD) and left-right diameters of SC and PC in these groups. Signal intensity and diameter of C7 spinal nerve were assessed on TIRM. RESULTS: Compared to control groups, signal intensities of DRG and PC were higher in SNN patients when using MEDIC, but the standardized diameters were shorter in either DRG or PC. Abnormal PC signal intensities were identified in eight out of nine SNN patients (89 %) with MEDIC and five out of nine (56 %) with T2-weighted images. SCA, assessed with MEDIC, was smaller in SNN patients than in the other groups, with significant reduction of its standardized APD. C7 nerve root diameters, assessed with TIRM, were decreased in SNN patients. CONCLUSION: MEDIC and TIRM sequences demonstrate increased signal intensities and decreased area of DRG and PC, and decreased diameter of nerve roots in patients with SNN, which can play a significant role in early diagnosis.
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Interpretación de Imagen Asistida por Computador/métodos , Imagen por Resonancia Magnética/métodos , Enfermedades del Sistema Nervioso Periférico/patología , Trastornos de la Sensación/patología , Adulto , Anciano , Plexo Braquial , Femenino , Ganglios Espinales , Humanos , Aumento de la Imagen/métodos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Médula Espinal , Adulto JovenRESUMEN
BACKGROUND: Thrombolytic treatment with intravenous (IV) recombinant tissue plasminogen activator (rtPA; 0.90 mg/kg, with a maximum dose of 90 mg) has been recommended as the standard management for acute ischemic stroke (AIS) thrombolysis. However, the dose of IV rtPA in Asia remains controversial. METHODS: This study was designed to verify the safety and efficacy of IV rtPA treatment for AIS with a lower dosage (0.90 mg/kg, with a maximum dose of 50 mg). Patients were divided into 3 dosage groups according to body weight (BW): group 1, <55 kg for 0.90 mg/kg; group 2, 55 to 67 kg for 0.75 to 0.90 mg/kg; and group 3, >67 kg for <0.75 mg/kg. The following data were collected: patient demographics, vascular risk factors, neuroimaging results, time of rtPA administration, National Institutes of Health Stroke Scale score before treatment and at 24 hours, and a modified Rankin Scale (mRS) score at 3 months. RESULTS: Eighty-three AIS patients who were of Han Chinese descent were included in the study. The baseline characteristics of the 3 dosage groups were well matched. In group 1 (BW <55 kg for 0.90 mg/kg; n = 19), 57.1% had a favorable outcome at 3 months, compared with 61.2% of patients in group 2 (BW 55-67 kg for 0.75-0.90 mg/kg; n = 33) and 51.5% in group 3 (BW >67 kg for <0.75 mg/kg; n = 31; P = .362). There were no significantly statistical differences in the incidence of symptomatic intracerebral hemorrhage and mortality rate. CONCLUSIONS: This IV rtPA regimen (0.90 mg/kg, with a maximum dose of 50 mg) not only shows sufficient favorable outcome in clinical practice in Chinese patients with AIS but also good health economic savings. This regimen could be suitable for many developing countries.
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Isquemia Encefálica/tratamiento farmacológico , Fibrinolíticos/administración & dosificación , Accidente Cerebrovascular/tratamiento farmacológico , Terapia Trombolítica , Activador de Tejido Plasminógeno/administración & dosificación , Anciano , Pueblo Asiatico , Isquemia Encefálica/diagnóstico , Isquemia Encefálica/etnología , Isquemia Encefálica/mortalidad , Hemorragia Cerebral/inducido químicamente , Distribución de Chi-Cuadrado , China/epidemiología , Evaluación de la Discapacidad , Femenino , Fibrinolíticos/efectos adversos , Humanos , Infusiones Intravenosas , Modelos Logísticos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Factores de Riesgo , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/etnología , Accidente Cerebrovascular/mortalidad , Terapia Trombolítica/efectos adversos , Terapia Trombolítica/mortalidad , Factores de Tiempo , Activador de Tejido Plasminógeno/efectos adversos , Resultado del TratamientoRESUMEN
Mitosis is a complicated and ordered process with high energy demands and metabolite fluxes. Cytosolic creatine kinase (CK), an enzyme involved in ATP homeostasis, has been shown to be essential to chromosome movement during mitotic anaphase in sea urchin. However, it remains elusive for the molecular mechanism underlying the recruitment of cytosolic CK by the mitotic apparatus. In this study, Fam96b/MIP18, a component of the MMXD complex with a function in Fe/S cluster supply, was identified as a brain-type CK (CKB)-binding protein. The binding of Fam96b with CKB was independent of the presence of CKB substrates and did not interfere with CKB activity. Fam96b was prone to oligomerize via the formation of intermolecular disulfide bonds, while the binding of enzymatically active CKB could modulate Fam96b oligomerization. Oligomerized Fam96b recruited CKB and the MMXD complex to associate with the mitotic spindle. Depletion of Fam96b or CKB by siRNA in the HeLa cells led to mitotic defects, which further resulted in retarded cell proliferation, increased cell death and aberrant cell cycle progression. Rescue experiments indicated that both Fam96b oligomerization and CKB activity were essential to the proper formation of mitotic spindle. These findings suggest that Fam96b may act as a scaffold protein to coordinate the supply and homeostasis of ATP and Fe/S clusters during mitosis.
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Creatina Quinasa , Huso Acromático , Humanos , Adenosina Trifosfato , Encéfalo/metabolismo , Creatina Quinasa/genética , Creatina Quinasa/metabolismo , Células HeLa , Huso Acromático/genética , Huso Acromático/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Early laboratory identification of group B Streptococcus (GBS, Streptococcus agalactiae) in the birth canal of pregnant women is critical for prompt administration of antimicrobial therapy and may further reduce the mortality rate due to GBS neonatal infection. METHODS: A total of 164 vaginal/rectal swab specimens collected from pregnant women at 35-37 weeks of gestation were screened for GBS vaginal colonization. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Biotyper, Bruker Daltonik GmbH, Bremen, Germany) system was used to detect GBS from Carrot broth and LIM broth enrichment using an in-house extraction protocol. The results were compared to those by conventional broth-enriched culture/identification methods as the gold standard. BD MAX™ GBS assay (Becton Dickinson, Sparks, MD, USA) was also performed for Carrot broth-enriched specimen. Discordant results were investigated using the GeneXpert® GBS PCR assay (Cepheid Inc., Sunnyvale, CA, USA). RESULTS: Using the extraction protocol, 33 (20.1%) of the 164 specimens were positive in Carrot broth, and 19 (11.6%) were positive in LIM broth. Using the culture protocol, 38 (23.2%) samples in Carrot broth and 35 (21.3%) in LIM broth were positive. The sensitivity, specificity, and positive and negative predictive values using the extraction protocol in Carrot broth and LIM broth compared to the gold standard conventional culture/identification method were 86.8% and 50.0%, 100% and 100%, 100% and 100%, and 96.2% and 86.9%, respectively. CONCLUSIONS: The extraction protocol with MALDI-TOF MS from Carrot broth-enriched samples provides a more rapid turnaround time, lower cost, and acceptable sensitivity and specificity to correctly identify pathogens when compared to conventional culture/identification methods.
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Infecciones Estreptocócicas , Streptococcus agalactiae , Recién Nacido , Embarazo , Femenino , Humanos , Recto , Vagina , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Rayos Láser , Infecciones Estreptocócicas/diagnósticoRESUMEN
Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.
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Forma MM de la Creatina-Quinasa/química , Forma MM de la Creatina-Quinasa/metabolismo , Animales , Forma MM de la Creatina-Quinasa/genética , Simulación de Dinámica Molecular , Mutación , Estructura Terciaria de Proteína , Desplegamiento Proteico , Conejos , TemperaturaRESUMEN
Heat shock protein 90 (Hsp90) is a molecular chaperone highly conserved across the species from prokaryotes to eukaryotes. Hsp90 is essential for cell viability under all growth conditions and is proposed to act as a hub of the signaling network and protein homeostasis of the eukaryotic cells. By interacting with various client proteins, Hsp90 is involved in diverse physiological processes such as signal transduction, cell mobility, heat shock response and osmotic stress response. In this research, we cloned the dshsp90 gene encoding a polypeptide composed of 696 amino acids from the halotolerant unicellular green algae Dunaliella salina. Sequence alignment indicated that DsHsp90 belonged to the cytosolic Hsp90A family. Further biophysical and biochemical studies of the recombinant protein revealed that DsHsp90 possessed ATPase activity and existed as a dimer with similar percentages of secondary structures to those well-studied Hsp90As. Analysis of the nucleotide sequence of the cloned genomic DNA fragment indicated that dshsp90 contained 21 exons interrupted by 20 introns, which is much more complicated than the other plant hsp90 genes. The promoter region of dshsp90 contained putative cis-acting stress responsive elements and binding sites of transcriptional factors that respond to heat shock and salt stress. Further experimental research confirmed that dshsp90 was upregulated quickly by heat and salt shock in the D. salina cells. These findings suggested that dshsp90 might serve as a component of the early response system of the D. salina cells against environmental stresses.
Asunto(s)
Chlorophyta/fisiología , Proteínas HSP90 de Choque Térmico/fisiología , Proteínas de Plantas/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/aislamiento & purificación , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Presión Osmótica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Regiones Promotoras Genéticas , Tolerancia a la Sal , Regulación hacia ArribaRESUMEN
BACKGROUND: Isolated sulfite oxidase deficiency (ISOD) caused by sulfite oxidase gene (SUOX) mutations is a rare neurometabolic disease associated with ectopia lentis (EL). However, few genotype-phenotype correlations have been established yet. METHODS: Potentially pathogenic SUOX mutations were screened from a Chinese cohort of congenital EL using panel-based next-generation sequencing and analyzed with multiple bioinformatics tools. The genotype-phenotype correlations were evaluated via a systematic review of SUOX mutations within our data and from the literature. RESULTS: A novel paternal missense mutation, c.205G > C (p.A69P), and a recurrent maternal nonsense mutation, c.1200 C > G (p.Y400*), of SUOX were identified in a 4-year-old boy from 312 probands. The biochemical assays manifested elevated urine sulfite and S-sulfocysteine accompanied by decreased homocysteine in the blood. The patient had bilateral EL and normal fundus, yet minimal neurological involvement and normal brain structure. Molecular modeling simulation revealed the p.A69P mutant had an unstable structure but an unchanged affinity for sulfite, while the truncated p.Y400* mutant showed decreased binding capacity. Genotype-phenotype analysis demonstrated patients with biallelic missense mutations had milder symptoms (P = 0.023), later age of onset (P < 0.001), and a higher incidence of regression (P = 0.017) than other genotypes. No correlations were found regarding EL and other neurological symptoms. CONCLUSION: The data from this study not only enrich the known mutation spectrum of SUOX but also suggest that missense mutations are associated with mild and atypical symptoms.