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991.
We demonstrate the formation of micropatterned sol-gel structures containing active proteins by patterning with polydimethylsiloxane (PDMS) microchannels. To transport sol solution efficiently into the hydrophobic PDMS microchannels, a hydrophilic-hydrophobic block copolymer was used to impart hydrophilicity to the PDMS microchannels. Poor adhesion of the micropatterned gel structure onto glass slides was improved by treating the glass surface with a polymeric substrate. To minimize cracks in the gel microstructure, hybrid matrices of interpenetrating organic and inorganic networks were prepared containing the reactive organic moieties polyvinylalcohol or polyvinylpyrrolidone. Retention of biochemical activity within the micropatterned gel was demonstrated by performing immunobinding assays with immobilized immunoglobulin G (IgG) antibody. The potential application of microfluidics technology to immobilized-enzyme biocatalysis was demonstrated using PDMS-patterned microchannels filled with trypsin-containing sol-gels. This work provides a foundation for the microfabrication of functional protein chips using sol-gel processes. 相似文献
992.
Habib AA Chatterjee S Park SK Ratan RR Lefebvre S Vartanian T 《The Journal of biological chemistry》2001,276(12):8865-8874
993.
Vibrio vulnificus cytolysin induces superoxide anion-initiated apoptotic signaling pathway in human ECV304 cells 总被引:10,自引:0,他引:10
Kwon KB Yang JY Ryu DG Rho HW Kim JS Park JW Kim HR Park BH 《The Journal of biological chemistry》2001,276(50):47518-47523
Previous studies showed that exposure to Vibrio vulnificus cytolysin (VVC) caused characteristic morphologic changes and dysfunction of vascular structures in lung. VVC showed cytotoxicity for mammalian cells in culture and acted as a vascular permeability factor. In this study, the underlying mechanisms of VVC-induced cytotoxicity was investigated on ECV304 cell, a human vascular endothelial cell line. When cells were exposed to 0.4 hemolytic units (HU) of VVC, consecutive apoptotic events were observed; the elevation of superoxide anion (O (-.)(2)), the release of cytochrome c, the activation of caspase-3, the cleavage of poly(ADP-ribose) polymerase, and the DNA fragmentation. The pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), O(-.) 2) scavenger, completely abolished O(-.)(2) levels and downstream apoptotic events. Moreover, pretreatment with cyclosporin A (CsA), a mitochondrial permeability transition inhibitor, was capable of attenuating O(-.)(2)-mediated cytochrome c release and caspase-3 activation, and consequent apoptosis. Apoptosis, as demonstrated by oligonucleosomal DNA fragmentation and fluorescence microscopy, was induced 24 h after VVC treatment, which was also prevented by caspase-3 inhibitor, Ac-DEVD-CHO. Caspase-1 inhibitor, Ac-YVAD-CHO, did not protect ECV 304 cells from apoptosis. These results suggest a scenario where VVC-induced apoptosis is triggered by the generation of O(-.)(2), release of cytochrome c from mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase, and DNA fragmentation. The induction of apoptosis in endothelial cells by VVC may provide a pivotal mechanism for understanding the pathophysiology of septicemia. 相似文献
994.
995.
996.
The expression of nitric oxide synthase (NOS) isoforms I, III and protein kinase-C (PKC) in rat vastus lateralis muscle was demonstrated immunohistochemically and then correlated to the physiological metabolic fibre types: SO (slow-oxidative), FOGI, FOGII (fast-oxidative glycolytic; I more glycolytic, II more oxidative), and FG (fast-glycolytic). NOS expression in muscles from different experimental groups (normal and diabetic rats, with and without Ginkgo biloba extract treatment) was assayed by Western blotting. Generally, NOS I and PKC were co-expressed in fibres with predominantly oxidative metabolism (SO, FOGII). This suggests an interplay of PKC and NOS I in nitric oxide production by oxidative fibres. NOS III was more highly expressed in fibres with predominantly glycolytic metabolism (FOGI, FG). A somewhat lower NOS I immunoreactivity was also found in NOS III positive fibres suggesting that NOS III and NOS I are co-expressed in these fibres. Western blotting revealed that NOS I as well as NOS III expression in the vastus lateralis muscle was down-regulated in diabetes and increased after Ginkgo biloba extract treatment. These effects may be associated with a diminished glucose uptake by myocytes of diabetic muscles and with an improved muscle function after Ginkgo biloba treatment. 相似文献
997.
Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene
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Johnson CL Pechonick E Park SD Havemann GD Leal NA Bobik TA 《Journal of bacteriology》2001,183(5):1577-1584
Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed. 相似文献
998.
Chung WO Park Y Lamont RJ McNab R Barbieri B Demuth DR 《Journal of bacteriology》2001,183(13):3903-3909
The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin. 相似文献
999.
The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA. 相似文献
1000.
Genetic and structural organization of the aminophenol catabolic operon and its implication for evolutionary process 总被引:3,自引:0,他引:3
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The aminophenol (AP) catabolic operon in Pseudomonas putida HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol meta cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene, nbzR, and the following nine structural genes, nbzJCaCbDGFEIH, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other meta cleavage operons led us to suggest that the regulatory unit (nbzR) was derived from the MarR family and that the structural unit (nbzJCaCbDGFEIH) has evolved from the ancestral meta cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer. 相似文献