Arabidopsis CSN5B Interacts with VTC1 and Modulates Ascorbic Acid Synthesis

Light regulates ascorbic acid (AsA) synthesis, which increases in the light, presumably reflecting a need for antioxidants to detoxify reactive molecules produced during photosynthesis. Here, we examine this regulation in Arabidopsis thaliana and find that alterations in the protein levels of the AsA biosynthetic enzyme GDP-Man pyrophosphorylase (VTC1) are associated with changes in AsA contents in light and darkness. To find regulatory factors involved in AsA synthesis, we identified VTC1-interacting proteins by yeast two-hybrid screening of a cDNA library from etiolated seedlings. This screen identified the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B), which interacted with the N terminus of VTC1 in yeast and plants. Gel filtration profiling showed that VTC1-CSN5B also associated with the COP9 signalosome complex, and this interaction promotes ubiquitination-dependent VTC1 degradation through the 26S proteasome pathway. Consistent with this, csn5b mutants showed very high AsA levels in both light and darkness. Also, a double mutant of csn5b with the partial loss-of-function mutant vtc1-1 contained AsA levels between those of vtc1-1 and csn5b, showing that CSN5B modulates AsA synthesis by affecting VTC1. In addition, the csn5b mutant showed higher tolerance to salt, indicating that CSN5B regulation of AsA synthesis affects the response to salt stress. Together, our data reveal a regulatory role of CSN5B in light-dark regulation of AsA synthesis.     

Hierarchically Clustering to 1,033 Genes Differentially Expressed in Mouse Superior Colliculus in the Courses of Optic Nerve Development and Injury

Tempo spatially specific expression of many development-related genes is the molecular basis for the formation of the central nervous system (CNS), especially those genes regulating the proliferation, differentiation, migration, axon growth, and orientation of nerve cells. The development-related genes are usually prominent during the embryonic and newborn stages, but rarely express during the adulthood. These genes are believed to be suitable target genes for promoting CNS regeneration, despite majority of which remains unknown. Hence, the aim of this study was to screen development-related genes which might contribute to CNS regeneration. In this study, 1,033 differentially-expressed genes of superior colliculus in the courses of mouse optic nerve development and injury, as previously identified by cDNA microarrays, were hierarchically clustered to display expression pattern of each gene and reveal the relationships among these genes, and infer the functions of some unknown genes based on function-identified genes with the similar expression patterns. Consequently, the expression patterns of 1,033 candidate genes were revealed at eight time points during optic nerve development or injury. According to the similarity among gene expression patterns, 1,033 genes were divided into seven groups. The potential function of genes in each group was inferred on the basis of the dynamic trend for mean gene expression values. Moreover, the expression patterns of six function-unidentified genes were extremely similar to that of the ptn gene which could promote and guide axonal extension. Therefore, these six genes are temporally regarded as candidate genes related to axon growth and guidance. The results may help to better understand the roles of function-identified genes in the stages of CNS development and injury, and offer useful clues to evaluate the functions of hundreds of unidentified genes.     

CAROTENOIDS PRODUCTION IN DIFFERENT CULTURE CONDITIONS BY Sporidiobolus pararoseus

Carotenoids produced by Sporidiobolus pararoseus were studied. It was found that biomass was connected with carbon source, temperature, and pH, but carotenoids proportion was seriously influenced by dissolved oxygen and nitrogen source. Different carotenoids could be obtained by using selected optimum conditions. In the end we established the strategies to produce β-carotene or torulene. Fed-batch fermentation in fermentor was used to prove the authenticity of our conclusions. The cell biomass, β-carotene content, and β-carotene proportion could reach 56.32 g/L, 18.92 mg/L and 60.43%, respectively, by using corn steep liquor at 0–5% of dissolved oxygen saturation. β-Carotene content was 271% higher than before this addition. The cell biomass, torulene content, and torulene proportion could reach 62.47 g/L, 31.74 mg/L, and 70.41%, respectively, by using yeast extract at 30–35% of dissolved oxygen saturation. Torulene content was 152% higher than before this addition. The strategy for enhancing specific carotenoid production by selected fermentation conditions may provide an alternative approach to enhance carotenoid production with other strains.     

PHOSPHONATE ANALOGS OF CARBOCYCLIC PHOSPHORIBOSYLAMINE AND CARBOCYCLIC GLYCINAMIDE RIBONUCLEOTIDE

Abstract

Analogs of intermediates in the de novo purine nucleotide biosynthetic pathway were synthesized to study the binding requirements of the corresponding enzymes. Because of the instability of the natural stubstrates, such as phosphoribosylamine, the use of the structurally stable phosphonate moiety and the carbocyclic ribose yields ideal analogs for these studies. In addition, these analogs can act as potential inhibitors of the de novo pathway leading to the design of anticancer agents. Enzyme studies with GAR synthetase and GAR transformylase reveal that the title compounds can act as substrates or inhibitors of the de novo enzymes.     

Metabolism of O6-Propyl and N6-Propyl-carbovir in CEM Cells

Abstract

The metabolism of O⁶-propyl-carbovir and N⁶-propyl-carbovir, two selective inhibitors of HIV replication, has been evaluated in CEM cells. Both compounds were phosphorylated in intact cells to carbovir-5′-triphosphate. The metabolism of these two agents was inhibited by deoxycoformycin and mycophenolic acid, but not erythro-9-(2-hydroxy-3-nonyl)adenine. No evidence of the 5′-triphosphate of either compound was detected in CEM cells.