Melanin biosynthesis in the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus)

Curvularia lunata (teleomorph: Cochliobolus lunatus) is a known plant and human pathogen. Tricyclazole, a specific inhibitor of pentaketide melanin biosynthesis, blocked the biosynthesis of melanin in Curvularia lunata and caused the accumulation of the melanin metabolites flaviolin and 2-hydroxyjuglone. This showed that melanin in Curvularia lunata is produced by a pentaketide pathway from 1,8-dihydroxynaphthalene. The 1,3,8-trihydroxynaphthalene reductase (3HNR) gene, associated with the melanin pathway of Curvularia lunata, was identified and characterized. An alignment of 3HNR sequences enabled the design of primers covering conserved regions. A PCR-amplified fragment of Curvularia lunata genomic DNA was used for screening the cDNA library. Three independent cDNA clones revealed an 801-bp open reading frame encoding a 267 amino acid protein. The protein was expressed in Escherichia coli and purified to homogeneity. The predicted amino acid sequence of the 28.6-kDa protein demonstrated homology to other fungal 3HNR and other members of the short-chain dehydrogenase super family. Northern analyses revealed that 3HNR from Curvularia lunata is expressed synchronously with melanization after 3 days of Curvularia lunata growth in malt extract medium. No 3HNR reductase gene expression nor melanization was observed when Curvularia lunata was grown in yeast nitrogen base medium.     

A proteomic study of the arabidopsis nuclear matrix

The eukaryotic nucleus has been proposed to be organized by two interdependent nucleoprotein structures, the DNA-based chromatin and the RNA-dependent nuclear matrix. The functional composition and molecular organization of the second component have not yet been resolved. Here, we describe the isolation of the nuclear matrix from the model plant Arabidopsis, its initial characterization by confocal and electron microscopy, and the identification of 36 proteins by mass spectrometry. Electron microscopy of resinless samples confirmed a structure very similar to that described for the animal nuclear matrix. Two-dimensional gel electrophoresis resolved approximately 300 protein spots. Proteins were identified in batches by ESI tandem mass spectrometry after resolution by 1D SDS-PAGE. Among the identified proteins were a number of demonstrated or predicted Arabidopsis homologs of nucleolar proteins such as IMP4, Nop56, Nop58, fibrillarins, nucleolin, as well as ribosomal components and a putative histone deacetylase. Others included homologs of eEF-1, HSP/HSC70, and DnaJ, which have also been identified in the nucleolus or nuclear matrix of human cells, as well as a number of novel proteins with unknown function. This study is the first proteomic approach towards the characterization of a higher plant nuclear matrix. It demonstrates the striking similarities both in structure and protein composition of the operationally defined nuclear matrix across kingdoms whose unicellular ancestors have separated more than one billion years ago.     

A fossil feather from the Upper Cretaceous of Kras (Slovenia)

A fossil feather preserved as a carbonised trace is described from the Tomaj Limestone at Kri , in the Kras region of southwestern Slovenia. The Tomaj Limestone is a platy and laminated limestone with cherts, which occurs within a well-bedded rudist limestone of the Santonian–Campanian Lipica Formation. It was deposited in a lagoon environment and has yielded a diverse fossil assemblage. Whether this feather belonged to a bird or to a dinosaur is unclear, but it is an addition to the scanty record of Late Cretaceous feathers, from a palaeobiogeographically interesting area.     

Arabidopsis WPP-domain proteins are developmentally associated with the nuclear envelope and promote cell division

The nuclear envelope (NE) acts as a selective barrier to macromolecule trafficking between the nucleus and the cytoplasm and undergoes a complex reorganization during mitosis. Different eukaryotic kingdoms show specializations in NE function and composition. In contrast with vertebrates, the protein composition of the NE and the function of NE proteins are barely understood in plants. MFP1 attachment factor 1 (MAF1) is a plant-specific NE-associated protein first identified in tomato (Lycopersicon esculentum). Here, we demonstrate that two Arabidopsis thaliana MAF1 homologs, WPP1 and WPP2, are associated with the NE specifically in undifferentiated cells of the root tip. Reentry into cell cycle after callus induction from differentiated root segments reprograms their NE association. Based on green fluorescent protein fusions and immunogold labeling data, the proteins are associated with the outer NE and the nuclear pores in interphase cells and with the immature cell plate during cytokinesis. RNA interference-based suppression of the Arabidopsis WPP family causes shorter primary roots, a reduced number of lateral roots, and reduced mitotic activity of the root meristem. Together, these data demonstrate the existence of regulated NE targeting in plants and identify a class of plant-specific NE proteins involved in mitotic activity.     

Cinnamic acid esters as potent inhibitors of fungal 17beta-hydroxysteroid dehydrogenase--a model enzyme of the short-chain dehydrogenase/reductase superfamily

We present the synthesis of a new family of nonsteroidal inhibitors of 17beta-hydroxysteroid dehydrogenase, designed from flavones and chalcones. Their inhibitory potential was screened on 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a model enzyme of the short-chain dehydrogenase/reductase superfamily. In a series of cinnamates and related coumarin-3-carboxylates, a number of compounds proved to be potent inhibitors of both the oxidative and reductive reactions catalyzed by 17beta-HSDcl, with IC(50) values in the low micromolar range.     

The PDZ-LIM protein RIL modulates actin stress fiber turnover and enhances the association of alpha-actinin with F-actin

ALP, CLP-36 and RIL form the ALP subfamily of PDZ-LIM proteins. ALP has been implicated in sarcomere function in muscle cells in association with alpha-actinin. The closely related CLP-36 is predominantly expressed in nonmuscle cells, where it localizes to actin stress fibers also in association with alpha-actinin. Here we have studied the expression and functions of RIL originally identified as a gene downregulated in H-ras-transformed cells. RIL was mostly expressed in nonmuscle epithelial cells with a pattern distinct from that of CLP-36. RIL protein was found to localize to actin stress fibers in nonmuscle cells similarly to CLP-36. However, RIL expression led to partially abnormal actin filaments showing thick irregular stress fibers not seen with CLP-36. Furthermore, live cell imaging demonstrated altered stress fiber dynamics with rapid formation of new fibers and frequent collapse of thick irregular fibers in EGFP-RIL-expressing cells. These effects may be mediated through the association of RIL with alpha-actinin, as RIL was found to associate with alpha-actinin via its PDZ domain, and RIL enhanced the ability of alpha-actinin to cosediment with actin filaments. These results implicate the RIL PDZ-LIM protein as a regulator of actin stress fiber turnover.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100 microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100 microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1 microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone all had IC(50) values between 1 and 5 microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

A QTL controlling stem morphology and vascular development in Lycopersicon esculentumxLycopersicon hirsutum (Solanaceae) crosses is located on chromosome 2

The vascular tissue of higher plants is organized into a continuous and unified system that undergoes a transition between two highly differentiated structures, the root and the shoot. This transition was studied in tomato by investigating the genetic basis of morphological variation between Lycopersicon esculentum and L. hirsutum LA407. Our analysis concentrated on morphology in stem cross sections, and we detected heritable genetic differences in an inbred backcross population having L. esculentum as the recurrent parent and LA407 as the donor parent. Inbred backcross line (IBL) 2353 contained a donor segment from chromosome 2 and retained features of the LA407 stem vascular morphology. Marker-trait analysis of vascular structure in a cross between IBL 2353 and L. esculentum showed significant (0.0001 ≤ P ≤ 0.0375) associations between markers on chromosome 2 and the size of primary vascular bundles, the shape of the vascular system, and the thickness of the secondary vascular tissue. Families with LA407 DNA for the markers on chromosome 2 had larger primary vascular bundles, more developed secondary vascular tissue, and a triangular vascular shape. These results suggest that the distal portion of chromosome 2 in LA407 contains a locus or loci affecting vascular morphology and development.     

Anchorage of plant RanGAP to the nuclear envelope involves novel nuclear-pore-associated proteins

The Ran GTPase controls multiple cellular processes including nucleocytoplasmic transport, spindle assembly, and nuclear envelope (NE) formation [1-4]. Its roles are accomplished by the asymmetric distribution of RanGTP and RanGDP enabled by the specific locations of the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1 [5-8]. Mammalian RanGAP1 targeting to the NE and kinetochores requires interaction of its sumoylated C-terminal domain with the nucleoporin Nup358/RanBP2 [9-14]. In contrast, Arabidopsis RanGAP1 is associated with the NE and cell plate, mediated by an N-terminal, plant-specific WPP domain [15-18]. In the absence of RanBP2 in plants, the mechanism for spatially sequestering plant RanGAP is unknown. Here, Arabidopsis WPP-domain interacting proteins (WIPs) that interact with RanGAP1 in vivo and colocalize with RanGAP1 at the NE and cell plate were identified. Immunogold labeling indicates that WIP1 is associated with the outer NE. In a wip1-1/wip2-1/wip3-1 triple mutant, RanGAP1 is dislocated from the NE in undifferentiated root-tip cells, whereas NE targeting in differentiated root cells and targeting to the cell plate remain intact. We propose that WIPs are novel plant nucleoporins involved in RanGAP1 NE anchoring in specific cell types. Our data support a separate evolution of RanGAP targeting mechanisms in different kingdoms.