domains (Hammond et al., 1995). In addition, PLD1 does and characterized. We find that a motif (HxKxxxxD) not encode sequences previously shown to interact with and a serine/threonine conserved in all members of RhoA (Reid et al., 1996). However, database searches the PLD superfamily are critical for PLD biochemical using mammalian PLD1 revealed the existence of PLD activity, suggesting a possible catalytic mechanism.homologs in numerous species (Hammond et al., 1995)

SUMMARYIn Arabidopsis, RNA editing changes more than 500 cytidines to uridines in mitochondrial transcripts. The editing enzyme and co-factors involved in these processes are largely unknown. We have identified a nuclear gene SLOW GROWTH1 (SLO1) encoding an E motif-containing pentatricopeptide repeat protein that is required for RNA editing of nad4 and nad9 in Arabidopsis mitochondria. The SLO1 protein is localized to the mitochondrion, and its absence gives rise to small plants with slow growth and delayed development. A survey of approximately 500 mitochondrial RNA editing sites in Arabidopsis reveals that the editing of two sites, nad4-449 and nad9-328, is abolished in the slo1 mutants. Sequence comparison in the upstream (from )1 to )15 bp) of nad4-449 and nad9-328 editing sites shows that nine of the 15 nucleotides are identical. In addition to RNA editing, we used RNA gel blot analysis to compare the abundance and banding patterns of mitochondrial transcripts between the wild type and slo1 mutants. Of the 79 genes and open reading frames examined, steady-state levels of 56 mitochondrial transcripts are increased in the slo1 mutants. These results suggest that the SLO1 protein may indirectly regulate plant growth and development via affecting mitochondrial RNA editing and gene expression.

RNA editing is one of the post-transcriptional processes that commonly occur in plant plastids and mitochondria. In Arabidopsis, 34 C-to-U RNA editing events, affecting transcripts of 18 plastid genes, have been identified. Here, we examined the editing and expression of these transcripts in different organs, and in green and non-green seedlings (etiolated, cia5-2, ispF and ispG albino mutants, lincomycin-, and norflurazon-treated). The editing efficiency of Arabidopsis plastid transcripts varies from site to site, and may be specifically regulated in different tissues. Steady state levels of plastid transcripts are low or undetectable in etiolated seedlings, but most editing sites are edited with efficiencies similar to those observed in green seedlings. By contrast, the editing of some sites is completely lost or significantly reduced in other non-green tissues; for instance, the editing of ndhB-149, ndhB-1255, and ndhD-2 is completely lost in roots and in lincomycin-treated seedlings. The editing of ndhD-2 is also completely lost in albino mutants and norflurazon-treated seedlings. However, matK-640 is completely edited, and accD-794, atpF-92, psbE-214, psbF-77, psbZ-50, and rps14-50 are completely or highly edited in both green and non-green tissues. In addition, the expression of nucleus-encoded RNA polymerase dependent transcripts is specifically induced by lincomycin, and the splicing of ndhB transcripts is significantly reduced in the albino mutants and inhibitor-treated seedlings. Our results indicate that plastid gene expression, and the splicing and editing of plastid transcripts are specifically and differentially regulated in various types of non-green tissues.

The vanilla cream1 (vac1) albino mutant is defective in a gene encoding a chloroplast-localized pentatricopeptide repeat protein of the DYW subgroup. However, the carboxyl-terminal DYW motif is truncated in VAC1. To identify vac1-specific phenotypes, we compared 34 chloroplast RNA editing sites and approximately 90 chloroplast gene expression patterns among wild type, vac1 and another albino mutant ispH, which is defective in the plastid isoprenoid biosynthesis pathway. We found that the editing of accD and ndhF transcripts is partially affected in vac1. In addition, steady-state levels of chloroplast rRNAs are significantly decreased in vac1. The expression of plastid-encoded RNA polymerase transcribed genes is down-regulated, whereas the expression of nucleus-encoded RNA polymerase transcribed genes is up-regulated in vac1. Although the development and function of mutant chloroplasts are severely impaired, steady-state mRNA levels of nucleus-encoded photosynthetic genes are not affected or are only slightly decreased in vac1. The ZAT10 gene encodes a transcription factor and its expression is down-regulated by norflurazon treatment in wild type. This norflurazon effect was not observed in vac1. These results suggest that the VAC1 protein may be involved in plastid-to-nucleus retrograde signaling in addition to its role in chloroplast RNA editing and gene expression. A defect in a key biosynthetic pathway can have many indirect effects on chloroplast gene expression as is seen in the ispH mutant. Similarly, the vac1 mutant has pleiotropic molecular phenotypes and most of which may be indirect effects.

BackgroundThe ACT domain, named after bacterial aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase), is a regulatory domain that serves as an amino acid-binding site in feedback-regulated amino acid metabolic enzymes. We have previously identified a novel type of ACT domain-containing protein family, the ACT domain repeat (ACR) protein family, in Arabidopsis. Members of the ACR family, ACR1 to ACR8, contain four copies of the ACT domain that extend throughout the entire polypeptide. Here, we describe the identification of four novel ACT domain-containing proteins, namely ACR9 to ACR12, in Arabidopsis. The ACR9 and ACR10 proteins contain three copies of the ACT domain, whereas the ACR11 and ACR12 proteins have a putative transit peptide followed by two copies of the ACT domain. The functions of these plant ACR proteins are largely unknown.ResultsThe ACR11 and ACR12 proteins are predicted to target to chloroplasts. We used protoplast transient expression assay to demonstrate that the Arabidopsis ACR11- and ACR12-green fluorescent fusion proteins are localized to the chloroplast. Analysis of an ACR11 promoter-β-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity was mainly detected in mature leaves and sepals. Interestingly, coexpression analysis revealed that the GLN2, which encodes a chloroplastic glutamine synthetase, has the highest mutual rank in the coexpressed gene network connected to ACR11. We used RNA gel blot analysis to confirm that the expression pattern of ACR11 is similar to that of GLN2 in various organs from 6-week-old Arabidopsis. Moreover, the expression of ACR11 and GLN2 is highly co-regulated by sucrose and light/dark treatments in 2-week-old Arabidopsis seedlings.ConclusionsThis study reports the identification of four novel ACT domain repeat proteins, ACR9 to ACR12, in Arabidopsis. The ACR11 and ACR12 proteins are localized to the chloroplast, and the expression of ACR11 and GLN2 is highly coordinated. These results suggest that the ACR11 and GLN2 genes may belong to the same functional module. The Arabidopsis ACR11 protein may function as a regulatory protein that is related to glutamine metabolism or signaling in the chloroplast.

The JAK2/STAT signaling pathway mediates cytokine receptor signals that are involved in cell growth, survival and homeostasis. JAK2 is a member of the Janus kinase (JAK) family and aberrant JAK2/STAT is involved with various diseases, making the pathway a therapeutic target. The similarity between the ATP binding site of protein kinases has made development of specific inhibitors difficult. Current JAK2 inhibitors are not selective and produce unwanted side effects. It is thought that increasing selectivity of kinase inhibitors may reduce the side effects seen with current treatment options. Thus, there is a great need for a selective JAK inhibitor. In this study, we identified a JAK2 specific inhibitor. We first identified key pharmacological interactions in the JAK2 binding site by analyzing known JAK2 inhibitors. Then, we performed structure-based virtual screening and filtered compounds based on their pharmacological interactions and identified compound NSC13626 as a potential JAK2 inhibitor. Results of enzymatic assays revealed that against a panel of kinases, compound NSC13626 is a JAK2 inhibitor and has high selectivity toward the JAK2 and JAK3 isozymes. Our cellular assays revealed that compound NSC13626 inhibits colorectal cancer cell (CRC) growth by downregulating phosphorylation of STAT3 and arresting the cell cycle in the S phase. Thus, we believe that compound NSC13626 has potential to be further optimized as a selective JAK2 drug.

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used sitedirected mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes.

Influenza is an annual seasonal epidemic that has continually drawn public attentions, due to the potential death toll and drug resistance. Neuraminidase, which is essential for the spread of influenza virus, has been regarded as a valid target for the treatment of influenza infection. Although neuraminidase drugs have been developed, they are susceptible to drug-resistant mutations in the sialic-binding site. In this study, we established computational models (site-moiety maps) of H1N1 and H5N1 to determine properties of the 150-cavity, which is adjacent to the drug-binding site. The models reveal that hydrogen-bonding interactions with residues R118, D151, and R156 and van der Waals interactions with residues Q136, D151, and T439 are important for identifying 150-cavitiy inhibitors. Based on the models, we discovered three new inhibitors with IC50 values <10 μM that occupies both the 150-cavity and sialic sites. The experimental results identified inhibitors with similar activities against both wild-type and dual H274Y/I222R mutant neuraminidases and showed little cytotoxic effects. Furthermore, we identified three new inhibitors situated at the sialic-binding site with inhibitory effects for normal neuraminidase, but lowered effects for mutant strains. The results suggest that the new inhibitors can be used as a starting point to combat drug-resistant strains.