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41.
Michele Michelin Vivian M. Benassi Luiz Alberto B. Moraes João A. Jorge Maria de Lourdes T.M. Polizeli 《Carbohydrate research》2010,345(16):2348-2353
An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t50 of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The Km of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase). 相似文献
42.
α‐ l‐iduronidase gene‐based therapy using the phiC31 system to treat mucopolysaccharidose type I mice 下载免费PDF全文
Roberta Sessa Stilhano Priscila Keiko Matsumoto Martin Suely Maymone de Melo Vivian Yochiko Samoto Giovani Bravin Peres Yara Maria Correa da Silva Michelacci Flavia Helena da Silva Vanessa Gonçalves Pereira Vania D'Almeida Adriana Taveira da Cruz Miriam Galvonas Jasiulionis Sang Won Han 《The journal of gene medicine》2015,17(1-2):1-13
43.
Zeng N Li Y He L Xu X Galicia V Deng C Stiles BL 《Molecular cancer research : MCR》2011,9(12):1708-1717
The α-subunit of eukaryotic initiation factor 2 (eIF2α) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2α phosphorylation can be regulated by a tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) and how such regulation is used by PTEN-deficient hepatocytes to adapt and cope with oxidative stress. We found that eIF2α was hyperphosphorylated when Pten was deleted, and this process was AKT dependent. Consistent with this finding, we found that the Pten-null cells developed resistance to oxidative glutamate and H(2)O(2)-induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2α phosphorylation), a constitutive phosphatase of eIF2α, was downregulated in Pten-null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2α phosphorylation. Ectopic expression of CReP restored the sensitivity of the Pten mutant hepatocytes to oxidative stress, confirming the functional significance of the downregulated CReP and upregulated phospho-eIF2α in the resistance of Pten mutant hepatocytes to cellular stress. In summary, our study suggested a novel role of PTEN in regulating stress response through modulating the CReP/eIF2α pathway. 相似文献
44.
45.
Toneff T Mende-Mueller L Wu Y Hwang SR Bundey R Thompson LM Chesselet MF Hook V 《Journal of neurochemistry》2002,82(1):84-92
Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD. 相似文献
46.
Andrade VS Rojas DB Oliveira L Nunes ML de Castro FL Garcia C Gemelli T de Andrade RB Wannmacher CM 《Molecular and cellular biochemistry》2012,362(1-2):225-232
It is known that the accumulation of tryptophan and its metabolites is related to brain damage associated with both hypertryptophanemia and neurodegenerative diseases. In this study, we investigated the effect of tryptophan administration on various parameters of behavior in the open-field task and oxidative stress, and the effects of creatine and pyruvate, on the effect of tryptophan. Forty, 60-day-old male Wistar rats, were randomly divided into four groups: saline, tryptophan, pyruvate + creatine, tryptophan + pyruvate + creatine. Animals received three subcutaneous injections of tryptophan (2 μmol/g body weight each one at 3 h of intervals) and/or pyruvate (200 μg/g body weight 1 h before tryptophan), and/or creatine (400 μg/g body weight twice a day for 5 days before tryptophan twice a day for 5 days before training); controls received saline solution (NaCl 0.85%) at the same volumes (30 μl/g body weight) than the other substances. Results showed that tryptophan increased the activity of the animals, suggesting a reduction in the ability of habituation to the environment. Tryptophan induced increase of TBA-RS and total sulfhydryls. The effects of tryptophan in the open field, and in oxidative stress were fully prevented by the combination of creatine plus pyruvate. In case these findings also occur in humans affected by hypertryptophanemia or other neurodegenerative disease in which tryptophan accumulates, it is feasible that oxidative stress may be involved in the mechanisms leading to the brain injury, suggesting that creatine and pyruvate supplementation could benefit patients affected by these disorders. 相似文献
47.
Hwang SR Garza C Mosier C Toneff T Wunderlich E Goldsmith P Hook V 《The Journal of biological chemistry》2007,282(13):9556-9563
Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides. 相似文献
48.
The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from
polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified
from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed
in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and
non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end
of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs
among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals.
Received February 17, 2000; accepted June 12, 2000. 相似文献
49.
The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles. 相似文献