Expression and purification of the recombinant mouse tumor supressor cytochrome b561 protein

It has recently been recognized that ascorbate-reducible cytochrome b561 (Cyt-b561) proteins constitute a well-distinguished protein family amongst the two-heme containing b-type cytochromes, ubiquitously present in animals and plants. Of the six isoforms that have been identified in mammals, three isoformes (called CGCytb, DCytb, and LCytb) have been cloned and expressed in yeast and/or bacterial cells. The recombinant proteins have been characterized in some detail. A particular gene product of the 3p21.3 (human) and 9F1 (mouse) chromosomal region, a so-called tumor supressor protein (101F6, TSP10), was identified as a Cyt-b561 protein by sequence homology. We have cloned and expressed the mouse tumor supressor Cyt-b561 protein (TSCytb) in yeast (Saccharomyces serevisiae), without and with a His6-tag on either the N- or the C-terminus. The C-terminal His6-tagged recombinant protein was purified on Ni-NTA His•Bind resin to almost homogeneity. Using optical spectroscopy we show that TSCytb is indeed an ASC-reducible cytochrome b561 protein and that ASC-reducibility is not affected by the presence of a His6-tag on the C-terminus. Minor differences in the properties of TSCytb and the other three mammalian Cyts-b561 are discussed

Ascorbate-reducible b-type cytochrome in the plant plasma membrane

It has been know for 20 years that the plasma membrane (PM) in plants contains more than one kind of ABSTRACT btype cytochromes. One of them has rather high redox potential (can fully be reduced by ascorbate) and is capable of electron transport through the PM. Three b-type cytochromes have recently been predicted from the full genome of Arabidopsis thaliana. In order to identify and characterize the one located in the PM, first PM vesicles were purified from Arabidopsis leaves, then the PM vesicles were solubilized and the fully ascorbate-reducible b-type cytochrome was partially purified. Redox titration of the partially purified b-type cytochrome revealed the presence of two hemes with redox potentials higher than 100 mV. The major polypeptide band of this fraction on SDS-PAGE was at ~120 kDa. This value is much higher than the apparent molecular mass of either the fully ascorbate-reducible b-type cytochrome purified from Phaseolus hypocotyls or the cyt. b-561 proteins purified from chromaffin granule membranes or the calculated molecular masses for the three polypeptides predicted from the full genome of Arabidopsis

Characterization of an ascorbate-reducible cytochrome b561 by site-directed mutagenesis

Ascorbate(ASC)-reducible cytochrome b561 (Cyt-b561) proteins are present in both plants and animals and create a well-distinguished protein family amongst the two-heme containing b-type cytochromes. One isoform of the Cyts-b561 identified by genomic analysis of Arabidopsis thaliana has been localized in the tonoplast. We have expressed the tonoplastlocalized Cyt-b561 (TCyt-b561) in yeast (Saccharomyces serevisiae) cells and shown that the biophysical properties of the recombinant TCyt-b561 is very similar to those of the chromaffin granule Cyt-b561 (CGCyt-b561). Mutation of 4 well-conserved histidine residues (H50, H83, H117, H156) resulted in different expression levels and revealed the importance of these 4 His residues in heme binding and protein expression. Modification of the protein by FLAG-tag or His6-tag resulted in different degrees of reduced expression levels. When all lysine residues (K70, K76, K79, K80, and K159) in the vicinity of the putative ASC-binding motive were one-by-one replaced by alanine, no major changes in the expression levels were observed. Except in case of the K80A mutant, where the low-affinity ASC-binding constant increased significantly, there were no significant changes in either kinetic parameter characterizing the bi-phase ASC-dependent reduction of TCytb-b561. These observations are discussed in comparison to properties of the recombinant CGCyt-b561

Substrate-dependent reduction of a recombinant chromaffin granule Cyt-b561 and its R72A mutant

Cytochrome b561 (Cyt-b561) proteins constitute a family of integral membrane proteins, catalyzing ASC-driven trans-membrane electron transport. Numerous isoforms of Cytb561 are present in invertebrates, vertebrates, and plants. The only protein of this family, however, which has been characterized in details at both biophysical, biochemical and physiological levels so far, is the bovine chromaffin granule Cyt-b561 (CGCyt-b561). Recently, both the bovine and the mouse CGCyt-b561 has been expressed in yeast cells and the recombinant proteins were shown to have biophysical properties similar to the native bovine CGCyt-b561. We have expressed the mouse CGCyt-b561 with a His6 -tag at the C terminus (CGCyt-b561(C6H)) in yeast (Saccharomyces serevisiae) cells and studied the reduction of CGCyt-b561(C6H) in the presence of different natural reducing agents. Besides the well-known natural reductant ascorbate (ASC) and the often-used artificial reductant dithionate, NADH, GSH, and dihydrolipoic acid (DHLA), also reduced the fully oxidized protein. Interestingly however, NADPH was not effective at all. When the same reductants were tested with the R72A mutant of CGCyt-b561(C6H), a mutant with impaired ASC-dependent reducibility, neither pyridine-dinucleotides could reduce the R72A mutant. DHLA-dependent and ASC-dependent reduction kinetics were very similar in case of the R72A mutant but differed in case of CGCyt-b561. These results raise the question of how many natural reductants the CGCyt-b561 may utilize in vivo

Oxidative stress biomarkers are associated with visible clinical signs of a disease in frigatebird nestlings

Infectious diseases are one of the most common threats for both domestic and wild animals, but little is known about the effects on the physiological condition and survival of wild animals. Here, we have tested for the first time in a wild vertebrate facing a viral disease possibly due to herpesvirus (i) whether nestlings with either low levels of oxidative damage or high levels of antioxidant protection are less susceptible to develop visible clinical signs, (ii) whether the disease is associated with the nestlings' oxidative status, (iii) whether the association between the disease and oxidative status is similar between males and females (iv), and whether cloacal and tracheal swabs might be used to detect herpesvirus. To address our questions, we took advantage of a population of Magnificent frigatebirds (Fregata magnificens) whose nestlings have experienced high mortality rates in recent times. Our work shows that (i) blood lipid oxidative damage is associated with observable clinical signs and survival probabilities of nestling frigatebirds, and (ii) that high glutathione levels in red blood cells are associated with the emergence of visible clinical signs of the disease. Our work provides evidence that differences in the oxidative status of nestlings might underlie individual health and survival

Drought induces distinct growth response, protection, and recovery mechanisms in the maize leaf growth zone

Drought is the most important crop yield-limiting factor, and detailed knowledge of its impact on plant growth regulation is crucial. The maize (Zea mays) leaf growth zone offers unique possibilities for studying the spatiotemporal regulation of developmental processes by transcriptional analyses and methods that require more material, such as metabolite and enzyme activity measurements. By means of a kinematic analysis, we show that drought inhibits maize leaf growth by inhibiting cell division in the meristem and cell expansion in the elongation zone. Through a microarray study, we observed the down- regulation of 32 of the 54 cell cycle genes, providing a basis for the inhibited cell division. We also found evidence for an up- regulation of the photosynthetic machinery and the antioxidant and redox systems. This was confirmed by increased chlorophyll content in mature cells and increased activity of antioxidant enzymes and metabolite levels across the growth zone, respectively. We demonstrate the functional significance of the identified transcriptional reprogramming by showing that increasing the antioxidant capacity in the proliferation zone, by overexpression of the Arabidopsis (Arabidopsis thaliana) iron-superoxide dismutase gene, increases leaf growth rate by stimulating cell division. We also show that the increased photosynthetic capacity leads to enhanced photosynthesis upon rewatering, facilitating the often-observed growth compensation.Fil: Avramova, Viktoriya. Universiteit Antwerp; BélgicaFil: Abdelgawad, Hamada. University of Beni-Suef; EgiptoFil: Zhang, Zhengfeng. Central China Normal University; ChinaFil: Fotschki, Bartosz. Institute of Animal Reproduction and Food Research; PoloniaFil: Casadevall, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; ArgentinaFil: Vergauwen, Lucia. Universiteit Antwerp; BélgicaFil: Knapen, Dries. Universiteit Antwerp; BélgicaFil: Taleisnik, Edith. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guisez, Yves. Universiteit Antwerp; BélgicaFil: Asard, Han. Universiteit Antwerp; BélgicaFil: Beemster, Gerrit T. S.. Universiteit Antwerp; Bélgic

Dihydrolipoic acid reduces cytochrome b561 proteins.

Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins

Vermicompost Supply Modifies Chemical Composition and Improves Nutritive and Medicinal Properties of Date Palm Fruits From Saudi Arabia

To meet the increased demand for phytochemicals, plant cultivation in soil amended with biofertilizers has been developed. Here, we aimed to use vermicompost as an environmentally safe biofertilizer to enhance the nutritive and medicinal value of five common cultivars of Saudi date palm; namely Phoenix dactylifera L. var. Ajwa, Hulwa, Ruthana, Sefri, and Luban. To determine changes in the fruit nutritive composition, primary metabolites, antioxidants, phenolic compounds and mineral profiles were analyzed in the fruits from non-fertilized and vermicompost-fertilized date palms. We also tested how changes in the fruit chemical compositions due to vermicompost fertilization affected their medicinal potentials. Applying vermicomposts generally increased primary metabolites, vitamins, and mineral content as well as the medicinal potential of the date palm fruits. This positive effect is possibly explained by the role of vermicomposts in improving soil health and fertility. Furthermore, clustering analyses and principal component analysis (PCA) indicated cultivar-specific responses. PCA analysis also revealed that the bioactivities of the date palm fruit extracts and their antioxidants tended to display correlated output values. One of the highly accumulated phenolic compounds, β-D-glucogallin, was extracted and purified from P. dactylifera L. var. Ajwa fruits and showed significant antioxidant, anticancer, antibacterial, antimutagenic, and antiprotozoal activities. Overall, applying vermicompost is an innovative approach to increase the nutritive quality and medicinal potential of date palm fruits

Brief Note on Ancient Paleobotany

International audienc

Relation of Plants to Food Science and Technology

International audienc