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The in vitro superoxide scavenging activity (as determined by electrochemical measurement) and the in vivo antioxidant potential (as determined by a mouse model of carbon tetrachloride (CCl4) hepatotoxicity) of methanolic extracts prepared from 10 Chinese tonifying herbs were compared. Electrochemical measurement using a cytochrome c (Cyt. c) sensor showed that all of the tested herbal extracts exhibited a medium superoxide scavenging activity of different potency, as indicated by their IC50 values. The in vivo measurement demonstrated that 80% of the herbal extracts displayed in vivo antioxidant potential, as assessed by the percentage of protection of the activity of plasma alanine aminotransferases and the hepatic glutathione regeneration capacity under CCl4-intoxicated condition. Although the in vitro antioxidant activity did not correlate quantitatively with the in vivo antioxidant potential, for 8 out of 10 samples a similar tendency was found. The rapid amperometric assessment of antioxidant potential by Cyt. c sensor may offer a convenient and direct method for screening as well as the quality control of herbal products. Copyright (C) 2004 John Wiley Sons, Ltd
The first IMPI (inhibitor of metalloproteinases from insects) was identified in the greater wax moth, Galleria mellonella [Wedde, Weise, Kopacek, Franke and Vilcinskas (1998) Eur. J. Biochem. 255, 535-543]. Here we report cloning and expression of a cDNA coding for this IMPI. The IMPI mRNA was identified among the induced transcripts from a subtractive and suppressive PCR analysis after bacterial challenge of G. mellonella larvae. Induced expression of the IMPI during a Immoral immune response was confirmed by real-time PCR, which documented up to 500 times higher amounts of IMPI mRNA in immunized larvae in comparison with untreated ones. The IMPI sequence shares no similarity with those of tissue inhibitors of metalloproteinases or other natural inhibitors of metalloproteinases, and the recombinant IMPI specifically inhibits thermolysin-like metalloproteinases, but not matrix metalloproteinases. These results support the hypothesis that the IMPI represents a novel type of immune-related protein which is induced and processed during the G. mellonella humoral immune response to inactivate pathogen-associated thermolysin-like metalloproteinases
The presence of partially folded intermediates along the folding funnel of proteins has been suggested to be a signature of potentially aggregating systems. Many studies have concluded that metastable, highly flexible intermediates are the basic elements of the aggregation process. In a previous paper, we demonstrated how the choice between aggregation and folding behavior was influenced by hydrophobicity distribution patterning along the sequence, as quantified by recurrence quantification analysis (RQA) of the Myiazawa-Jernigan coded primary structures. In the present paper, we tried to unify the "partially folded intermediate" and "hydrophobicity/charge" models of protein aggregation verifying the ability of an empirical relation, developed for rationalizing the effect of different mutations on aggregation propensity of acyl-phosphatase and based on the combination of hydrophobicity RQA and charge descriptors, to discriminate in a statistically significant way two different protein populations: (a) proteins that fold by a process passing by partially folded intermediates and (b) proteins that do not present partially folded intermediates
Glucose dehydrogenase (GDH) was assembled electrostatically onto QCM-gold electrodes by their sequential deposition with anionic polyelectrolytes such as PSS and PASA. For the layer-by-layer arrangements both the microgravimetric and the electrochemical sensor signal were followed. Increasing amounts of GDH were deposited by stepwise formation of alternating layers of GDH and PSS or PASA. The mass increase was about 1.88 mug/cm(2) for one GDH/ PASA bilayer and 2.4 mug/cm(2) for a GDH/PSS bilayer. The addition of phenolic compounds resulted in an oxidation current, which could be catalytically increased by the GDH catalysed reaction in the presence of glucose. The system functions as glucose sensor when quinones are present in nonlimiting amount. The amperometric response was already diffusion limited when a single layer of GDH was adsorbed. The sensor sensitivity increased by a factor of 10 when MSA was used instead of MUA as initial electrode modifier
Biosensorik / Bioanalytik
(2004)
Beweidung mit Wildtieren
(2004)
Beweidung mit Haustieren
(2004)
Benefit of regular Exercise on Cardiovascular Risk Factors and Motor Development in Early Childhood
(2004)
The Arabidopsis tandem-pore K+ (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K+ channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K+ currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K+ transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K+ channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode similar to55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K-m and V-max values were 125 muM for DAG and 0.25 pmol of PA min(-1) mug(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg2+-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca2+. AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2- dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4degreesC), pointing to a role in cold signal transduction
SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K+ channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K-out channels. Deletion mutants and chimeric proteins generated from SKOR and the K-in channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains thatchannel a-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K-T domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K-out alpha-subunits did not assemble with K-in alpha-subunits because of the absence of interaction between their assembly sites