Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase

The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro. Further modification of its C-2 substituent has led to the discovery of RO-09-4879, which exhibits antifungal activity in vivo. The drug design is based on X-ray crystal analysis of a CaNmt complex with benzofuran derivative 4a. The optimization incorporates various biological investigations including a quasi in vivo assay and pharmacokinetic study. The computer aided drug design, synthesis, structure-activity relationships, and biological properties of RO-09-4879 are described in detail.     

Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level

Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the protoplasts could be induced rapidly and specifically by co-transfection with the reporter gene and various silencing inducers [i.e. siRNA, double-stranded RNA (dsRNA) or plasmid encoding dsRNA]. Using this system, we uncovered novel roles of some viral suppressors. Notably, the Cucumber mosaic virus 2b protein, shown previously to function predominantly by preventing the long-distance transmission of systemic silencing signals, was a very strong silencing suppressor in the protoplasts. Some suppressors thought to interfere with upstream steps of siRNA production appeared to also act downstream. Therefore, a viral suppressor can affect multiple steps of the RNA silencing pathway. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells.     

Relationship between G1287A of the NET Gene Polymorphisms and Brain Volume in Major Depressive Disorder: A Voxel-Based MRI Study

Background

Earlier studies implicated norepinephrine transporter (NET) gene (SLC6A2) polymorphisms in the etiology of major depressive disorder (MDD). Recently, two single nucleotide SLC6A2 polymorphisms, G1287A in exon 9 and T-182C in the promoter region, were found to be associated with MDD in different populations. We investigated the relationship between the brain volume and these two polymorphisms of the SLC6A2 in MDD patients.

Methods

We obtained 3D high-resolution T1-weighted images of 30 first-episode MDD patients and 48 age- and sex-matched healthy subjects (HS). All were divided into 4 groups based on polymorphism of either the G1287A or the T-182C genotype. VBM analysis examined the effects of diagnosis, genotype, and genotype-diagnosis interactions.

Results

Diagnosis effects on the brain morphology were found in the left superior temporal cortex. No significant genotype effects were found in the T-182C and the G1287A. A significant genotype (G1287A)–diagnosis interaction was found in the left dorsolateral prefrontal cortex. No significant genotype (T-182C)–diagnosis interaction effects were observed in any brain region.

Conclusions

In MDD patients there seems to be a relationship between the volume of the dorsolateral prefrontal cortex and polymorphism of the SLC6A2 G1287A gene.     

Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family

Phospholipase A1 (PLA1) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids and is conserved in a wide range of organisms. Mammals have several enzymes that exhibit PLA1 activity in vitro. The extracellular PLA1s include phosphatidylserine (PS)-specific PLA1 (PS-PLA1), membrane-associated phosphatidic acid (PA)-selective PLA1s (mPA-PLA1alpha and mPA-PLA1beta), hepatic lipase (HL), endothelial lipase (EL) and pancreatic lipase-related protein 2 (PLRP2), all of which belong to the pancreatic lipase gene family. The former three PLA1s differ from other members in their substrate specificities, structural features and gene organizations, and form a subfamily in the pancreatic lipase gene family. PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta exhibit only PLA1 activity, while HL, EL and PLRP2 show triacylglycerol-hydrolyzing activity in addition to PLA1 activity. The tertiary structures of lipases have two surface loops, the lid and the beta9 loop. The lid and the beta9 loop cover the active site in its closed conformation. An alignment of amino acid sequences of the pancreatic lipase gene family members revealed two molecular characteristics of PLA1s in the two surface loops. First, lipase members exhibiting PLA1 activity (PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta, EL, guinea pig PLRP2 and PLA1 from hornet venom (DolmI)) have short lids. Second, PS-PLA1, mPA-PLA1alpha, mPA-PLA1beta and DolmI, which exhibit only PLA(1) activity, have short beta9 loops. Thus, the two surface loops appear to be involved in the ligand recognition. PS-PLA1 and mPA-PLA1s specifically hydrolyze PS and PA, respectively, producing their corresponding lysophospholipids. Lysophosphatidylserine and lysophosphatidic acid have been defined as lipid mediators with multiple biological functions. Thus, these PLA1s have a role in the production of these lysophospholipid mediators.     

Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55 degrees C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.     

The promoter of rbcS in a C3 plant (rice) directs organ-specific,light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding -glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.     

EpCAM contributes to formation of functional tight junction in the intestinal epithelium by recruiting claudin proteins

Tight junctions (TJs) connect epithelial cells and form a semipermeable barrier that only allows selective passage of ions and solutes across epithelia. Here we show that mice lacking EpCAM, a putative cell adhesion protein frequently overexpressed in human cancers, manifest intestinal barrier defects and die shortly after birth as a result of intestinal erosion. EpCAM was found to be highly expressed in the developing intestinal epithelium of wild-type mice and to localize to cell-cell junctions including TJs. Claudin-7 colocalized with EpCAM at cell-cell junctions, and the two proteins were found to associate with each other. Claudins 2, 3, 7, and 15 were down-regulated in the intestine of EpCAM mutant mice, with claudin-7 being reduced to undetectable levels. TJs in the mutant intestinal epithelium were morphologically abnormal with the network of TJ strands scattered and dispersed. Finally, the barrier function of the intestinal epithelium was impaired in the mutant animals. These results suggest that EpCAM contributes to formation of intestinal barrier by recruiting claudins to cell-cell junctions.     

The expression of tobacco knotted1-type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model

We have isolated and characterized four tobacco homeobox genes, NTH1, NTH9, NTH20, NTH22 (Nicotiana tabacum homeobox) which belong to the class 1 knotted1-type family of homeobox genes. Comparison of the inferred amino acid sequences of the ELK homeodomains of these genes and previously reported kn1-type class 1 proteins has revealed that the four new tobacco genes belong to distinct subclasses, suggesting that each NTH gene may have distinct functions. Using in situ hybridization and by analysing the distribution of GUS activity in tobacco plants transformed with NTH promoter::GUS constructs, localized expression of the three NTH genes was observed in the shoot apical meristem (SAM). In the vegetative SAM, NTH1 and NTH15 showed overlapping expression in the corpus, NTH20 was expressed in the peripheral zone, and NTH9 was predominantly expressed in the rib zone. The expression patterns of the different NTH genes correspond to regions predicted by the cytohistological zonation model, suggesting that each NTH gene specifies the function of the SAM zone with which it is associated.     

Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions

The SGS1 gene of Saccharomyces cerevisiae is a homologue for the Bloom's syndrome and Werner's syndrome genes. The disruption of the SGS1 gene resulted in very poor sporulation, and the majority of the cells were arrested at the mononucleated stage. The recombination frequency measured by a return-to-growth assay was reduced considerably in sgs1 disruptants. However, double-strand break formation, which is a key event in the initiation of meiotic DNA recombination, occurred; crossover and noncrossover products were observed in the disruptants, although the amounts of these products were slightly decreased compared with those in wild-type cells. The spores produced by sgs1 disruptants showed relatively high viability. The sgs1 spo13 double disruptants sporulated poorly, like the sgs1 disruptants, but spore viability was reduced much more than with either sgs1 or spo13 single disruptants. Disruption of the RED1 or RAD17 gene partially alleviated the poor-sporulation phenotype of sgs1 disruptants, indicating that portions of the population of sgs1 disruptants are blocked by the meiotic checkpoint. The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants.