Improved expression of novel red- and green-emitting luciferases of Phrixothrix railroad worms in mammalian cells.
(49/900)
Luciferases are widely used for the quantitative monitoring of gene expression in a variety of organisms. We successfully expressed novel red- and green-emitting luciferases of Phrixothrix railroad worms in mammalian cells in combination with the Kozak sequence and the CAG promoter. The characteristic properties of these luciferases indicate that they are appropriate reporter genes for the simultaneous monitoring of two gene expressions. (+info)
Bidirectional role of orphan nuclear receptor RORalpha in clock gene transcriptions demonstrated by a novel reporter assay system.
(50/900)
Circadian rhythms are generated by an extremely complicated transcription-translation feedback loop. To precisely analyze the molecular mechanisms of the circadian clock, it is critical to monitor multiple gene expressions and/or interactions with their transcription factors simultaneously. We have developed a novel reporter assay system, the tricolor reporter in vitro assay system, which consists of green- and red-emitting Phrixothrix luciferases as dual reporters and blue-emitting Renilla luciferase as internal control. We have successfully employed this system in analyzing the effects of clock gene products on the enhancer elements of Per1 and Bmal1 promoters. The results indicate that the orphan nuclear receptor RORalpha regulates bidirectionally Bmal1 (positively) and Per1 (negatively) transcriptions simultaneously. (+info)
Zymosan-induced luminol-dependent chemiluminescence response of circulating and extravasated leukocytes in experimental sepsis.
(51/900)
This study examines a concurrent profiling of circulating and extravasated polymorphonuclear leukocytes (PMNs) in a rat model of experimental sepsis. Fecal peritonitis was induced in Wistar male rats by intraperitoneal instillation of a fecal suspension in saline (1:1 w/v). Blood and peritoneal fluid were collected 8 h following fecal inoculation for the evaluation of inflammatory response of PMNs using zymosan-induced luminol-dependent chemiluminescence. Fifty microliters of pre-diluted blood or peritoneal fluid samples were mixed with 150 microl of reaction mixture (4 x 10(-4) M luminol+50 microg opsonized zymosan+0.1% gelatin in Hank's balanced salt solution) and the chemiluminescence signal was measured in a luminometer at 37 degrees C. Fecal peritonitis caused a significant leukocytopenia (3540+/-297 mm(-3) versus control value of 7525+/-711 mm(-3), p < 0.001) accompanied by massive infiltration of PMNs in the peritoneal cavity (34700+/-4006 versus 7325+/-425 mm(-3), p < 0.001). The phagocytic activity of circulating blood PMNs was down-regulated whereas a significant up-regulation was observed in the activity of PMNs from peritoneal fluid. In conclusion, this study clearly demonstrates sepsis-induced alterations in both blood and peritoneal fluid PMNs and their quantitative assessment may be helpful in disease evaluation and designing effective therapies. (+info)
Hypochlorous acid inhibition by acetoacetate: implications on neutrophil functions.
(52/900)
Type-1 diabetic patients experience hyperketonemia caused by an increase in fatty acid metabolism. Thus, the aim of this study was to measure the effect of ketone bodies as suppressors of oxidizing species produced by stimulated neutrophils. Both acetoacetate and 3-hydroxybutyrate have suppressive effect on the respiratory burst measured by luminol-enhanced chemiluminescence. Through measurements of hypochlorous acid production, using neutrophils or the myeloperoxidase/H2O2/Cl- system, it was found that acetoacetate but not 3-hydroxybutyrate is able to inhibit the generation of this antimicrobial oxidant. The superoxide anion scavenging properties were confirmed by ferricytochrome C reduction and lucigenin-enhanced chemiluminescence assays. However, ketone bodies did not alter the rate of oxygen uptake by stimulated neutrophils, measured with an oxygen electrode. A strong inhibition of the expression of the cytokine IL-8 by cultured neutrophils was also observed; this is discussed with reference to the antioxidant-like property of acetoacetate. (+info)
Antioxidant potential of Anogeissus latifolia.
(53/900)
Anogeissus latifolia is widely used in the Indian indigenous system of medicine and is reported to contain leucocyanidins and tannoid principles like ellagic acid and its derivatives. In view of its wide use and its chemical composition, this study was aimed at examining the antioxidant activity of the extract of A. latifolia. The extract was studied for total antioxidant capacity, hydrogen-donating ability, nitric oxide, superoxide scavenging activity, hydrogen peroxide decomposition activity along with lipid peroxidation. Integral antioxidative capacity was determined by chemiluminescence assay. The extract was also studied for lipid peroxidation assay by thiobarbituric acid-reactive substances (TBARS) method using rat liver homogenate. The results indicate that A. latifolia extract has potent antioxidant activity. Also to ascertain the possible reason for the potent activity, percentage of gallic acid was estimated and was found to be 0.95%, which could be one of the reasons for potent antioxidant activity exhibited by the plant. (+info)
Random mutagenesis of bacterial luciferase: critical role of Glu175 in the control of luminescence decay.
(54/900)
Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as Photobacterium fisheri, P. phosphoreum and P. leiognathi, have rapid decay rates. By substitution of a 67-amino-acid stretch of P. phosphoreum LuxA in the central region of the LuxA subunit, the 'slow' X. luminescens luciferase was converted into a chimaeric luciferase with a significantly more rapid decay rate [Valkova, Szittner and Meighen (1999) Biochemistry 38, 13820-13828]. To understand better the role of specific residues in the classification of luciferases as slow and fast decay, we have conducted random mutagenesis on this region. One of the mutants generated by a single mutation on LuxA at position 175 [E175G (Glu175-->Gly)] resulted in the 'slow decay' X. luminescens luciferase being converted into a luciferase with a significantly more rapid decay rate. These results indicate the importance of Glu175 in LuxA as a critical residue for differentiating between 'slow' and 'fast' luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate. (+info)
Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC.
(55/900)
Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria. (+info)
Thermoluminescence evidence for light-induced oxidation of tyrosine and histidine residues in manganese-depleted photosystem II particles.
(56/900)
In the thermoluminescence (TL) glow curve of photosystem II, particles depleted of manganese, a tyrosine modifier, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) abolishes the TL band appearing around -55 degrees C (TL-55). Addition of a histidine modifier, diethylpyrocarbonate results in the disappearance of the band peaking around -30 degrees C (TL-30). NBD treatment also abolishes the EPR signal IIfast of oxidized tyrosine donor, Yz, and inhibits the electron transport from diphenylcarbazide to 2,6-dichlorophenol-indophenol. It is concluded that the TL-55 and TL-30 bands can be assigned to oxidized tyrosine (Yz+) and histidine (His+) residues, respectively, which participate in electron transfer from manganese to the reaction center of chlorophyll, P680+. (+info)