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Biochim Biophys Acta Gen Subj ; 1861(11 Pt A): 2717-2725, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28754385


BACKGROUND: Cellular dynamics depend on cytoskeletal filaments and motor proteins. Collective movements of filaments driven by motor proteins are observed in the presence of dense filaments in in vitro systems. As multiple macromolecules exist within cells and the physiological ionic conditions affect their interactions, crowding might contribute to ordered cytoskeletal architecture because of collective behavior. METHODS: Using an in vitro reconstituted system, we observed the emergence of stripe patterns resulting from collective actin filament streaming driven by myosin motors in the presence of the crowding agent, methylcellulose (MC). RESULTS: Although at high KCl concentrations (150mM), actin filaments tended to dissociate from a myosin-coated surface, 1% MC prevented this dissociation and enabled filament movement on myosin molecules. At concentrations of actin filaments above 0.2mg/mL, the moving filaments accumulated and progressively formed long, dense bands. The bands were spaced at about 10-µm intervals. Increasing the KCl concentration up to 300mM resulted in narrowing of the spacing between the aligned bands. On the other hand, low KCl concentrations (≤25mM) induced broad streams, where actin filaments exhibited bidirectional movement. CONCLUSIONS: These results suggest that crowded environments can promote spatial patterning of the actin cytoskeleton, depending on the intensity of the myosin driving force and filament velocity, both modulated by the ionic strength. GENERAL SIGNIFICANCE: The mutual contribution of packing and driving forces provides insight into cytoskeleton organization in living cells, in which various macromolecules mingle.

Citoesqueleto de Actina/química , Actinas/química , Metilcelulose/química , Miosinas/química , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/química , Animais , Citoesqueleto/química , Citoesqueleto/metabolismo , Meio Ambiente , Metilcelulose/metabolismo , Movimento/efeitos dos fármacos , Miosinas/metabolismo , Cloreto de Potássio/química
Int J Mol Sci ; 14(2): 2590-600, 2013 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-23358249


We have previously reported that pyrroloquinoline quinone (PQQ) prevents the amyloid formation of α-synuclein, amyloid ß(1-42) (Aß(1-42)), and mouse prion protein. Moreover, PQQ-modified α-synuclein and a proteolytic fragment of the PQQ-modified α-synuclein are able to inhibit the amyloid formation of α-synuclein. Here, we identified the peptide sequences that play an important role as PQQ-modified specific peptide inhibitors of α-synuclein. We demonstrate that the PQQ-modified α-Syn(36-46) peptide, which is a partial sequence of α-synuclein, prevented α-synuclein amyloid fibril formation but did not inhibit Aß(1-42) fibril formation. In addition, the α-synuclein partial peptide modified with other small-molecule inhibitors, Baicalein and epigallocatechin gallate (EGCG), prevented α-synuclein fibril formation. Currently reported quinone amyloid inhibitors do not have selectivity toward protein molecules. Therefore, our achievements provide a novel strategy for the development of targeted specific amyloid formation inhibitors: the combination of quinone compounds with specific peptide sequence from target proteins involved in amyloid formation.

Anal Chem ; 85(1): 185-92, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23145996


The growing interest in membrane interactions of amyloidogenic proteins indicates that lipid binding and the regulation of membrane potential are critical to the onset and progression of neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and prion diseases. Advancing the understanding of this field requires the application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. Therefore, the development of a rapid cytotoxicity evaluation system using a membrane potential-sensitive bis-oxonol fluorescent dye, DiBAC4(3) is reported here. The exposure of C-terminal truncated α-synuclein 119 (α-Syn119) and amyloid-ß(1-42) (Aß(1-42)) to U2-OS cell cultures resulted in an immediate, significant, and concentration-dependent increase in fluorescence response of DiBAC4(3). This response was strongly correlated with the cytotoxicity of α-Syn119 and Aß(1-42) as determined by conventional CC8 and ATP assays. Furthermore, the capacity of well-defined polyphenolic antioxidants (i.e., pyrroloquinoline quinone (PQQ), baicalein, (-)-epigallocatechin-3-gallate (EGCG), and myricetin) to mitigate amyloid-induced cytotoxicity was evaluated using the developed biosensing system. We envisage that this work would accelerate the development of a rapid and cost-effective high-throughput screening platform in drug discovery for AD and PD.

Peptídeos beta-Amiloides/antagonistas & inibidores , Técnicas Biossensoriais , Corantes Fluorescentes/química , Fragmentos de Peptídeos/antagonistas & inibidores , alfa-Sinucleína/antagonistas & inibidores , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/química , Barbitúricos/química , Catequina/análogos & derivados , Catequina/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavanonas/química , Humanos , Isoxazóis/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/toxicidade , Tiobarbitúricos/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
Biosens Bioelectron ; 24(11): 3299-305, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19450964


A semi-automated bacterial spore detection system (BSDS) was developed to detect biological threat agents (e.g., Bacillus anthracis) on-site. The system comprised an aerosol sampler, micro-fluidic chip-A (for spore germination and cell lysis), micro-fluidic chip-B (for extraction and detection of genomic DNA) and an analyzer. An aerosol with bacterial spores was first collected in the collection chamber of chip-A with a velocity of 300 l/min, and the chip-A was taken off from the aerosol sampler and loaded into the analyzer. Reagents packaged in the chip-A were sequentially applied into the chamber. The genomic DNA extract from spore lyzate was manually transferred from chip-A to chip-B and loaded into the analyzer. Genomic DNA in chip-B was first trapped on a glass bead column, washed with various reagents, and eluted to the detection chamber by sequential auto-dispensing. Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) with fluorescent measurement was adopted to amplify and detect target DNA. Bacillus subtilis was the stimulant of biological warfare agent in this experiment. Pretreatment conditions were optimized by examining bacterial target DNA recovery in the respective steps (aerosol collection, spore germination, cell lysis, and DNA extraction), by an off-chip experiment using a real-time polymerase chain reaction quantification method. Without the germination step, B. subtilis spores did not demonstrate amplification of target DNA. The detection of 10(4) spores was achieved within 2h throughout the micro-fluidic process.

Aerossóis/análise , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Técnicas Biossensoriais/instrumentação , DNA Bacteriano/análise , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Microbiologia do Ar , Algoritmos , DNA Bacteriano/genética , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Técnicas de Amplificação de Ácido Nucleico/métodos , Esporos Bacterianos/genética , Esporos Bacterianos/isolamento & purificação
Intern Med ; 44(9): 975-8, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16258215


A 44-year-old woman on maintenance hemodialysis was admitted to our hospital because of severe abdominal pain. The patient had been medicated with lisinopril and valsartan for hypertension for one month prior to admission. An abdominal computerized scan (CT) showed a dilated and thickened loop of the small bowel with massive ascites and a small nodule in the jejunum. The patient's abdominal pain was thought to be due to isolated visceral angioneurotic edema induced by lisinopril and/or valsartan, and medication of these two drugs was therefore stopped. Her symptoms resolved and an abdominal CT demonstrated almost complete resolution of ascites and of small bowel edema except for a small nodule in the jejunum. A laparoscopic operation was performed to excise the small nodule of the jejunum, and a histological diagnosis of accessory pancreas of the jejunum was made. This is the first report of isolated visceral angioneurotic edema induced by lisinopril and/or valsartan in a patient on maintenance hemodialysis and, moreover, with the association of accessory pancreas of the jejunum.

Angioedema/induzido quimicamente , Anti-Hipertensivos/efeitos adversos , Lisinopril/efeitos adversos , Tetrazóis/efeitos adversos , Valina/análogos & derivados , Adulto , Angioedema/diagnóstico , Bloqueadores do Receptor Tipo 1 de Angiotensina II/efeitos adversos , Inibidores da Enzima Conversora de Angiotensina/efeitos adversos , Diagnóstico Diferencial , Feminino , Glomerulonefrite Membranoproliferativa/tratamento farmacológico , Glomerulonefrite Membranoproliferativa/terapia , Humanos , Diálise Renal , Valina/efeitos adversos , Valsartana