The use of phenylmethylsulfonyl fluoride in the study of catabolite inactivation and repression in intact cells of Saccharomyces cerevisiae

Catabolite inactivation of fructose-1,6-bisphosphatase, isocitrate lyase, phosphoenolpruvate carboxykinase and malate dehydrogenase in intact cells could be prevented by phenylmethylsulfonyl fluoride added 40 min prior to the addition of glucose. Protein synthesis, fermentative and respiratory activity and catabolite repression were not affected. Elimination of catabolite inactivation by the addition of PMSF revealed that catabolite repression started at different times for different enzyme.Abbreviation PMSF phenylmethylsulfonyl fluoride     

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5? were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532–587?nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.     

Nitric Oxide Interferes with Islet Cell Zinc Homeostasis

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function.     

Proteins of the sieve-tube exudate of Cucurbita maxima

Summary Sieve-tube exudate which appears on cut surfaces of stems of Cucurbita maxima as distinct droplets has been depicted in electron micrographs of longitudinal sections of the phloem. The exudate, which was produced from mature sieve tubes only, contained filaments of P-protein, but no mitochondria or vesicles of endoplasmic reticulum. The water-soluble part of the exudate contained at least 12 proteins, as shown by disc-electrophoresis. Enzymic activity was found for peroxidases, acid phosphatases, and aldolases. Color tests and assays for other enzymes, including ATPase, fructokinase, several dehydrogenases, and UDP-glucose: D-fructose-2-glucosyl transferase, gave negative results. With repeated cutting of a stem, the protein content of the exudate increased, while the amount of exudate decreased.Supported by Deutsche Forschungsgemeinschaft and Stiftung Volkswagenwerk. During part of this investigation the senior author held a U.S. National Science Foundation Senior Foreign Scientist Fellowship at the University of Wisconsin.     

A high density physical map of chromosome 1BL supports evolutionary studies,map-based cloning and sequencing in wheat

Background

As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.

Results

Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.

Conclusions

Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.     

Quinone binding and reduction by respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron–sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal β-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox–Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.     

Novel fluorescent core-shell nanocontainers for cell membrane transport

The synthesis and characterization of novel core-shell macromolecules consisting of a fluorescent perylene-3,4,9,10-tetracarboxdiimide chromophore in the center surrounded by a hydrophobic polyphenylene shell as a first and a flexible hydrophilic polymer shell as a second layer was presented. Following this strategy, several macromolecules bearing varying polymer chain lengths, different polymer shell densities, and increasing numbers of positive and negative charges were achieved. Because all of these macromolecules reveal a good water solubility, their ability to cross cellular membranes was investigated. In this way, a qualitative relationship between the molecular architecture of these macromolecules and the biological response was established.