RESUMEN
The fruit of Terminalia chebula Retz has been used as a traditional medicine in Asia and contains tannic acid, chebulagic acid, chebulinic acid and corilagin. Extract from T. chebula seeds (TCE) has various biological functions. We observed the neuroprotective effects of TCE against ischemic damage in the hippocampal C1 region (CA1) of the gerbil that had received oral administrations of TCE (100 mg/kg) once a day for 7 days before the induction of transient cerebral ischemia. In the TCE-treated ischemia group, neuronal neuclei (a marker for neurons)-positive neurons were distinctively abundant (62% of the sham group) in the CA1 4 days after ischemia-reperfusion (I-R) compared to those (12.2% of the sham group) in the vehicle-treated ischemia group. Four days after I-R TCE treatment markedly decreased the activation of astrocytes and microglia in the ischemic CA1 compared with the vehicle-treated ischemia group. In addition, immunoreactivities of Cu, Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2) and brain-derived neurotrophic factor (BDNF) in the CA1 of the TCE-treated ischemia group were much higher than those in the vehicle-ischemia group 4 days after I-R. Protein levels of SOD1, SOD2 and BDNF in the TCE-treated ischemia group were also much higher than those in the vehicle-ischemia group 4 days after I-R. These results indicate that the repeated supplement of TCE protected neurons from ischemic damage induced by transient cerebral ischemia by maintaining SODs and BDNF levels as well as decreasing glial activation.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Región CA1 Hipocampal/efectos de los fármacos , Ataque Isquémico Transitorio/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Fitoterapia , Extractos Vegetales/uso terapéutico , Superóxido Dismutasa/metabolismo , Terminalia/química , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Gerbillinae , Ataque Isquémico Transitorio/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismoRESUMEN
In this study, the function of nitric oxide (NO) in endoplasmic reticulum (ER)-related cell death in human glioma cells was investigated. Treatment of human CRT-MG cells with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and thapsigargin, an ER stress inducer, increased cytosolic Ca(2+) and caused apoptosis in a dose-dependent manner. Expression of the ER-associated molecules inositol-requiring enzyme 1 (IRE1)-alpha, p-eIF, and Ero1-alpha were also elevated in thapsigargin- or NO donor-treated cells. Furthermore, thapsigargin and SNAP treatment increased IRE1-alpha nuclease activity, induced IRE1-alpha/TRAF2 complex formation, and increased p-JNK1/2 levels, suggesting that NO activates the IRE1-alpha/TRAF2/JNK pathway in the ER. Expression of IRE1-alpha increased concomitantly with cAMP responsive element binding protein (CREB) phosphorylation. siRNA knock down of IRE1-alpha reduced phospho-CREB levels and abolished its nuclear translocation. The levels of phospho-CREB and IRE1-alpha increased with NO donor concentration, which resulted in cell death. IRE1-alpha and phospho-CREB levels in glioblastoma U87MG cells were higher than those in normal astrocytes in response to NO. In addition, treatment with the intracellular cytokine interleukin-1beta induced cell death associated with NO and increased IRE1-alpha and p-CREB levels. These data reveal that intracellular NO affects IRE1-alpha-dependent CREB phosphorylation in human glioma cells. Therefore, an IRE1-alpha-dependent phospho-CREB signaling pathway responsive to NO/Ca(2+) may play an important role in regulating ER-related cell death in glioma.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Retículo Endoplásmico/metabolismo , Endorribonucleasas/metabolismo , Glioma/metabolismo , Glioma/patología , Óxido Nítrico/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , S-Nitroso-N-Acetilpenicilamina/farmacología , Tapsigargina/farmacologíaRESUMEN
In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the 32P-labeled partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the Cys90, His226, and Asn250 residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3% to 12.5% of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and 35 degrees, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Escherichia coli/genética , Poliquetos/enzimología , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Catepsina F , Catepsinas/química , Catepsinas/genética , Catepsinas/metabolismo , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , ADN Complementario/química , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Fibrina/metabolismo , Regulación Enzimológica de la Expresión Génica , Biblioteca de Genes , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Poliquetos/genética , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.
Asunto(s)
Clonación Molecular , Escherichia coli/enzimología , Ferritinas/genética , Ferritinas/metabolismo , Poliquetos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Escherichia coli/genética , Ferritinas/química , Biblioteca de Genes , Humanos , Proteínas Reguladoras del Hierro/química , Proteínas Reguladoras del Hierro/metabolismo , Corea (Geográfico) , Ratones , Datos de Secuencia Molecular , Poliquetos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
We report upon the purification and characterization of a novel prothrombin-activating enzyme from the body fluid (total homogenates of isolated digestive tract without eggs, spinnerets and silk glands) of the spider, Nephila clavata by a combination of acetone fractionation, ion exchange, and Soybean trypsin inhibitor-Sepharose chromatography. Analysis of the purified enzyme with SDS-PAGE and gel filtration revealed a single polypeptide chain with an apparent molecular weight of 24kDa. The proteolytic activity of the enzyme was stable up to 50 degrees C, however, it became unstable over 55 degrees C. The enzyme had an optimum pH of 8, and Ca(2+) was not required for the enzyme activity. According to inhibition profiles obtained with several serine protease inhibitors such as PMSF and benzamidine, the purified protease is a member of the serine proteases. Bz-Ile-Glu(gamma-OR)- Gly-Arg-pNA and Z-Arg-Gly-Arg-pNA which are known as substrates for factor Xa, were hydrolyzed favorably by the enzyme. And the Nephila protease could produce thrombin from prothrombin at nM range, and form the turbid ring using fibrinogen-agarose plate. The results obtained confirmed that the purified protease is a potent prothrombin-activating activity belonging to the family of serine protease.
Asunto(s)
Protrombina/metabolismo , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Arañas/química , Trombina/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Peso Molecular , Proteínas/metabolismo , Serina Endopeptidasas/químicaRESUMEN
An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; K(2)HPO(4), 2; KH(2)PO(4), 2; maltose, 20; sodium citrate, 10; MgSO(4), 0.06; NaCl, 8; and Na(2)CO(3), 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CMSepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The K(m) and k(cat) values were 78.7 µM and 217.4 s(-1) for N-succinyl-Ala- Ala-Pro-Phe-pNA at 37° C and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and H(2)O(2), has significance for industrial application, such as additives in detergent and feed industries.