An aflatoxin biosynthesis cluster gene encodes a novel oxidase required for conversion of versicolorin a to sterigmatocystin

Disruption of the aflatoxin biosynthesis cluster gene aflY (hypA) gave Aspergillus parasiticus transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.     

β₂ long-acting and anticholinergic drugs control TGF-β1-mediated neutrophilic inflammation in COPD

We quantified TGF-β1 and acetylcholine (ACh) concentrations in induced sputum supernatants (ISSs) from 18 healthy controls (HC), 22 healthy smokers (HS) and 21 COPDs. ISSs from HC, HS and COPD as well as rhTGF-β1 were also tested in neutrophil adhesion and in mAChR2, mAChR3 and ChAT expression experiments in human bronchial epithelial cells (16-HBE). Finally, we evaluated the effects of Olodaterol (a novel inhaled β(2)-adrenoceptor agonist) and Tiotropium Spiriva?, alone or in combination, on neutrophil adhesion and mAChRs and ChAT expression in stimulated 16-HBE. The results showed that 1) TGF-β1 and ACh concentrations are increased in ISSs from COPD in comparison to HC and HS, and TGF-β1 in HS is higher than in HC; 2) ISSs from COPD and HS caused increased neutrophil adhesion to 16-HBE when compared to ISSs from HC. The effect of ISSs from COPD was significantly reduced by TGF-β1 depletion or by the pretreatment with Olodaterol or Tiotropium alone or in combination, while the effect of ISSs from HS was significantly reduced by the pretreatment with Olodaterol alone; 3) mAChR2, mAChR3 and ChAT expression was increased in 16-HBE stimulated with ISSs from COPD and TGF-β1 depletion significantly reduced this effect on mAChR3 and ChAT expression; 4) rhTGF-β1 increased mAChR2, mAChR3 and ChAT expression in 16-HBE; 5) Olodaterol did not affect the expression of mAChRs and ChAT in 16-HBE. Our findings support the use of β? long-acting and anticholinergic drugs to control the bronchoconstriction and TGF-β1-mediated neutrophilic inflammation in COPD.     

Unequal VH gene rearrangement frequency within the large VH7183 gene family is not due to recombination signal sequence variation, and mapping of the genes shows a bias of rearrangement based on chromosomal location.

Much of the nonrandom usage of V, D, and J genes in the Ab repertoire is due to different frequencies with which gene segments undergo V(D)J rearrangement. The recombination signal sequences flanking each segment are seldom identical with consensus sequences, and this natural variation in recombination signal sequence (RSS) accounts for some differences in rearrangement frequencies in vivo. Here, we have sequenced the RSS of 19 individual V(H)7183 genes, revealing that the majority have one of two closely related RSS. One group has a consensus heptamer, and the other has a nonconsensus heptamer. In vitro recombination substrate studies show that the RSS with the nonconsensus heptamer, which include the frequently rearranging 81X, rearrange less well than the RSS with the consensus heptamer. Although 81X differs from the other 7183-I genes at three positions in the spacer, this does not significantly increase its recombination potency in vitro. The rearrangement frequency of all members of the family was determined in microMT mice, and there was no correlation between the in vitro recombination potential and V(H) gene rearrangement frequency in vivo. Furthermore, genes with identical RSS rearrange at different frequencies in vivo. This demonstrates that other factors can override differences in RSS potency in vivo. We have also determined the gene order of all V(H)7183 genes in a bacterial artificial chromosome contig and show that most of the frequently rearranging genes are in the 3' half of the region. This suggests that chromosomal location plays an important role in nonrandom rearrangement of the V(H)7183 genes.     

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.      

Arabidopsis At5g39790 encodes a chloroplast-localized,carbohydrate-binding,coiled-coil domain-containing putative scaffold protein

Background  

Starch accumulation and degradation in chloroplasts is accomplished by a suite of over 30 enzymes. Recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, and the action of associated non-enzymatic regulatory proteins. Arabidopsis At5g39790 encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain.     

Identification and Characterization of RBEL1 Subfamily of GTPases in the Ras Superfamily Involved in Cell Growth Regulation

Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236–302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.The Ras superfamily is known to comprise five structurally distinct subfamilies of small GTPases, including Ras, Rho, Rab, Sar1/Arf, and Ran, and each subfamily of these GTPases possess distinct functions in the regulation of a variety of cellular processes such as cell proliferation, cell differentiation, cytoskeletal organization, protein transport, and trafficking (1–4). The Ras subfamily of GTPases (N-, H-, and K-Ras) function predominantly in relaying signals from receptors at the plasma membrane and modulating cell signaling pathways that regulate cell proliferation, differentiation, and survival (5). Ran GTPase, on other hand, is a key regulator of nucleocytoplasmic transport that regulates protein transport across the nuclear pore complex (6, 7). The Rab subfamily is the largest subfamily among the Ras superfamily and contains more than 60 members. The key functions of the Rab GTPases are to regulate protein exocytic and endocytic pathways and modulate intracellular protein transport/trafficking (8–13).In general, the Ras superfamily GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. There are five N-terminal motifs involved in the binding and hydrolysis of GTP that are highly conserved among all GTPases: G1 (GXXXXGK(S/T)), G2 (T), G3 (DXXG), G4 ((N/T)(K/Q)XD), and G5 (EXSAX). Each sequence has particular functions involved in binding nucleotides (GTP or GDP) and facilitating hydrolysis (4, 14, 15). In general, the intrinsic GTPase activity (converting GTP to GDP) and exchange of GDP for GTP are slow processes for these GTPases and thus require regulatory proteins such as GTPase-activating proteins and GDP/GTP exchange factors to facilitate these processes (16–18).For the last two decades, the Ras superfamily has been a major focus in the cancer field as many of the members are either mutated or dysregulated in cancer. The founding members of the Ras superfamily, H-Ras and K-Ras, were first identified as viral oncogenes (1, 4). Later studies demonstrated that mutations of the Ras proteins (H-, N-, and K-Ras) occur frequently in human cancers, and the mutations identified are mostly clustered within the GTP-binding domains of the proteins thus locking Ras proteins in a GTP-bound configuration. GTP-bound Ras is constitutively active; it constantly activates its effector proteins to transduce cell proliferative signals (1, 4). Unlike Ras subfamily genes, mutations occurring in Rab and Rab-like genes are less common, yet alterations in gene expression of a number of Rab genes have been reported in multiple human malignancies. For example, Rab25 overexpression has been linked to prostate cancer progression (19). Rab2 overexpression has been found in lung adenomas and adenocarcinomas (20). In addition, alterations in Rab gene expression have also been linked to cancer drug resistance. For instance, resistance to the anticancer drug doxorubicin in MCF-7 cells has been linked with reduced expression of Rab6C, and introduction of exogenous Rab6C restores drug sensitivity (21).We have recently reported the identification two novel Ras superfamily GTPases, RBEL1A and RBEL1B (22). RBEL1A and RBEL1B are two splice variants of the RBEL1 gene and are highly homologous to the Rab and Ran GTPases within their N-terminal GTP-binding domains (22). Our studies show that both RBEL1A and -B predominantly bind to GTP. A single point mutation (T57N) in the GTP-binding domain of RBEL1A and -B abolishes their ability to bind to both GTP and GDP. Both RBEL1A and RBEL1B localize in the nucleus as well as in the cytosol. Whereas RBEL1A is predominantly cytosolic, RBEL1B is primarily nuclear. Interestingly, our studies also suggested that nucleotide (GTP or GDP)-binding could be important for the nuclear distribution of RBEL1B, because the nucleotide binding-deficient mutant form (T57N) of RBEL1B did not reside in the nucleus but rather became largely cytosolic (22).In our continuous efforts to fully elucidate the function of RBEL1, we have identified two additional splice variants that we have named RBEL1C and RBEL1D. Here we report further characterization of all four RBEL1 splice variants in terms of their GTPase activities, subcellular localizations, regulations, and potential functions. Our results indicate that RBEL1 GTPases, although sharing some common features with other Ras superfamily members, also harbor unique characteristics that are significantly different from other Ras superfamily GTPases. Based on our findings, we suggest that RBEL1 proteins appear to form a novel subfamily of GTPases within the Ras superfamily.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

Synthesis of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and no DNA damage

In the search for new photochemotherapeutic agents, a series of derivatives of the ring system pyrrolo[3,2-h]quinoline—bioisosters of the angular furocoumarin angelicin—were synthesized through a four-step synthetic approach, in reasonable overall yields. Eight of the synthesized derivatives showed a remarkable phototoxicity against a panel of four human tumor cell lines and a great dose UV-A dependence, reaching IC₅₀ values at submicromolar level. The mode of cellular death photoinduced by pyrrolo[3,2-h]quinolines was evaluated through a series of flow cytometric analysis and other tests were performed to clarify their mechanism of action.