Chemosensitization of Aflatoxigenic Fungi to Antimycin A and Strobilurin Using Salicylaldehyde,a Volatile Natural Compound Targeting Cellular Antioxidation System

Various species of fungi in the genus Aspergillus are the most common causative agents of invasive aspergillosis and/or producers of hepato-carcinogenic mycotoxins. Salicylaldehyde (SA), a volatile natural compound, exhibited potent antifungal and anti-mycotoxigenic activities to A. flavus and A. parasiticus. By exposure to the volatilized SA, the growth of A. parasiticus was inhibited up to 10–75% at 9.5 mM ≤ SA ≤ 16.0 mM, while complete growth inhibition was achieved at 19.0 mM ≤ SA. Similar trends were also observed with A. flavus. The aflatoxin production, i.e., aflatoxin B₁ and B₂ (AFB₁, AFB₂) for A. flavus and AFB₁, AFB₂, AFG₁, and AFG₂ for A. parasiticus, in the SA-treated (9.5 mM) fungi was reduced by ~13–45% compared with the untreated control. Using gene deletion mutants of the model yeast Saccharomyces cerevisiae, we identified the fungal antioxidation system as the molecular target of SA, where sod1Δ [cytosolic superoxide dismutase (SOD)], sod2Δ (mitochondrial SOD), and glr1Δ (glutathione reductase) mutants showed increased sensitivity to this compound. Also sensitive was the gene deletion mutant, vph2Δ, for the vacuolar ATPase assembly protein, suggesting vacuolar detoxification plays an important role for fungal tolerance to SA. In chemosensitization experiments, co-application of SA with either antimycin A or strobilurin (inhibitors of mitochondrial respiration) resulted in complete growth inhibition of Aspergillus at much lower dose treatment of either agent, alone. Therefore, SA can enhance antifungal activity of commercial antifungal agents required to achieve effective control. SA is a potent antifungal and anti-aflatoxigenic volatile that may have some practical application as a fumigant.     

Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans

Background

Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans.

Methodology/Principal Findings

Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression.

Conclusions/Significance

We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes.     

Cells by Design: A Mini-Review of Targeting Cell Engineering Using DNA Microarrays

Recent studies have demonstrated the utility of DNA microarray technology in engineering cellular properties. For instance, cellular adhesion, the necessity of cells to attach to a surface in order to to proliferate, was examined by comparing two distinct HeLa cell lines. Two genes, one encoding a type II membrane glycosylating sialyltransferase (siat7e) and the other encoding a secreted glycoprotein (lama4), were found to influence adhesion. The expression of siat7e correlated with reduced adhesion, whereas expression of lama4 correlated with increased adhesion, as shown by various assays. In a separate example, a gene encoding a mitochondrial assembly protein (cox15) and a gene encoding a kinase (cdkl3), were found to influence cellular growth. Enhanced expression of either gene resulted in slightly higher specific growth rates and higher maximum cell densities for HeLa, HEK-293, and CHO cell lines. Another investigated property was the adaptation of HEK-293 cells to serum-free media. The genes egr1 and gas6, both with anti-apoptotic properties, were identified as potentially improving adaptability by impacting viability at low serum levels. In trying to control apoptosis, researchers found that by altering the expression levels of four genes faim, fadd, alg-2, and requiem, apoptotic response could be altered. In the present work, these and related studies in microorganisms (prokaryote and eukaryote) are examined in greater detail focusing on the approach of using DNA microarrays to direct cellular behavior by targeting select genes.     

Targeting radiosensitizers to DNA by attachment of an intercalating group: nitroimidazole-linked phenanthridines

The nitroimidazole-linked phenanthridine series of compounds (NLP-1, 2, and 3) were synthesized under the assumption that it should be possible to enhance the molar efficiency of 2-nitroimidazoles as hypoxic cell radiosensitizers and cytotoxins by targeting them to their likely site of action, DNA. The targeting group chosen was the phenanthridine moiety, the major component of the classical DNA intercalating compound, ethidium bromide. The sole difference between the compounds is the length of the hydrocarbon chain linking the nitroimidazole to the phenanthridine. The phenanthridine group with a three-carbon side chain, P-1, was also synthesized to allow studies on the effect of the targeting group by itself. The ability of the compounds to bind to DNA is inversely proportional to their linker chain length with binding constant values ranging from approximately 1 x 10(5) mol-1 for NLP-2 to 6 x 10(5) mol-1 for NLP-3. The NLP compounds show selective toxicity to hypoxic cells at 37 degrees C at external drug concentrations 10-40 times lower than would be required for untargeted 2-nitroimidazoles such as misonidazole in vitro. Toxicity to both hypoxic and aerobic cells is dependent on the linker chain: the shorter the chain, the greater the toxicity. In addition, the NLP compounds radiosensitize hypoxic cells at external drug concentrations as low as 0.05 mM with almost the full oxygen effect being observed at a concentration of 0.5 mM. These concentrations are 10-100 times lower than would be required for similar radiosensitization using misonidazole. Radiosensitizing ability is independent of linker chain length. The present compounds represent prototypes for further studies of the efficacy and mechanism of action of 2-nitroimidazoles targeted to DNA by linkage to an intercalating group.     

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.     

Locating Interrupted Hydrogen Bonding in the Secondary Structure of PM2 Circular DNA by Comparative Denaturation Mapping

Previous studies with HCHO have revealed a reaction with superhelical DNA that strongly suggests that this DNA consists of small regions of interrupted secondary structure. To map these sites in PM2 DNA, the following set of experiments was performed using electron microscopy. (i) A denaturation map of nicked form II was obtained using Inman's alkaline-HCHO conditions. (ii) The superhelical form I was reacted with HCHO at 30 C until equilibrium was achieved at the interrupted sites (3.6% reactivity). The excess HCHO was removed rapidly and X-ray treatment was employed to nick these prereacted molecules. These form II molecules containing HCHO (form II HCHO) were also subjected to denaturation mapping. It would be expected that the HCHO-unpaired regions would serve as induction sites for the propagation of melting. Hence, depending on the location of the induction sites; we would anticipate either the creation of new regions of melting or a normal denaturation map shifted to lower pH values. Comparison of the development of progressive denaturation of form II and form II HCHO reveals that the latter is the case. The denaturation maps of form II are highly organized patterns of adenine-thymine (AT)-rich regions, with a total of five regions at extreme pH conditions. There are six highly organized regions for form II HCHO, i.e., smaller adjacent loops, at low denaturation conditions where no denaturation is seen for form II. These coalesce into the pattern for form II containing four of five A-T-rich regions observed for form II. Hence we conclude that the regions of altered hydrogen bonding in superhelical PM2 DNA are four to six in number and they map in the A-T-rich regions of the DNA.     

Targeting of inositol 1,4,5-trisphosphate receptors to the endoplasmic reticulum by multiple signals within their transmembrane domains

Most inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in the endoplasmic reticulum (ER), where their precise distribution underlies the spatially complex Ca2+ signals evoked by extracellular stimuli. The signals that target IP3R to the ER or, less commonly, to other membranes are unknown. We expressed yellow fluorescent protein-tagged fragments of type 1 IP3R alone or fused with a plasma membrane protein to establish the determinants of ER targeting in COS-7 cells. By using a combination of confocal imaging and glycoprotein analyses, we demonstrated that any pair of the six transmembrane domains (TMD) linked by a luminal loop retains the protein within the ER, and when attached to a plasma membrane protein (ICAM-1), prevents it from reaching the medial Golgi. TMD1 or TMD2 alone were accumulated in mitochondria, whereas TMD5 and TMD6 were retained in ER, but were unable to prevent ICAM from reaching the plasma membrane. We conclude that IP3R are targeted to the ER membrane only after synthesis of TMDs 1 and 2, and that after co-translational insertion of the remaining TMDs, redundant retention signals present in any pair of TMD retain IP3R in the ER.     

Structural Characterization of a DNA Duplex Modeled on a DNA:RNA Hybrid of the Polypurine Tract Recognized by a Reverse Transcriptase

Abstract

The structure of d(TTAAAAGAAAAGGG):d(CCCTTTTCTTTTAA) has been characterized by NMR. The minor grooves of the two dA-tracts are suggested to be rather narrow, and the portion linking the two dA tracts exhibits a slightly deviated structure from a standard B DNA, in order to maintain the narrowness of the minor groove. The structure of the dG-tract is also slightly deviated. Additionally, specific broadening of resonances is observed for the residues at or near the junction between the dA-tract and the dG-tract, suggesting local structural polymorphology.

     

Differential Scanning Calorimetry (DSC): A Tool to Study the Thermal Behavior of Lipid Bilayers and Liposomal Stability

Thermodynamical techniques are applied for determining the thermal stress of medicinal compounds of the excipients as well as their interactions during the formulation process.

The physicochemical properties and the stability of the medicinal products could be measured as a function of temperature or time using thermal analysis.

Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for determining the purity, the polymorphic forms and the melting point of a sample in the Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring thermodynamic parameters (i.e. ΔH and Tm), which affect the stability of the liposomal suspension under given storage conditions.     

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

The removal of initiating primers from the 5′-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.     

A Note on the Detection of Phytopathogenic Bacteria within the Leaf by a Differential Staining Procedure

A simple differential staining procedure for demonstrating infection within the leaf tissue by Pseudomonas tomato, Ps. lachrymans and Xanthomonas vesicatoria has been developed. It is based on (1) clearing of plant tissue with a mixture of glycerol, lactic acid, phenol and water; (2) treating the leaf tissue with boiling KOH; and (3) staining with aniline blue-chloralhydrate. When observed under a light microscope, the bacteria appear dark blue, whereas the leaf tissue appears transparent and colourless.     

Loss of a DNA binding site within the tail of prelamin A contributes to altered heterochromatin anchorage by progerin

Mutations in the lamin A/C (LMNA) gene that cause Hutchinson-Gilford progeria syndrome (HGPS) lead to expression of a protein called progerin with 50 amino acids deleted from the tail of prelamin A. In cells from patients with HGPS, both the amount and distribution of heterochromatin are altered. We designed in vitro assays to ask whether such alterations might reflect changes in chromatin, DNA and/or histone binding properties of progerin compared to wild-type lamin C-terminal tails. We show that progerin tail has a reduced DNA/chromatin binding capacity and modified trimethylated H3K27 binding pattern, offering a molecular mechanism for heterochromatin alterations related to HGPS.

Structured summary

MINT-7893924, MINT-7893941, MINT-7893990, MINT-7894005, MINT-7894023, MINT-7894038: H3 (uniprotkb:Q71DI3) binds (MI:0407) to LaminA (uniprotkb:P02545) by surface plasmon resonance (MI:0107)MINT-7893957, MINT-7893974, MINT-7894055: H3 (uniprotkb:Q71DI3) binds (MI:0407) to progerin (uniprotkb:Q6UYC3) by surface plasmon resonance (MI:0107)     

ThioFinder: A Web-Based Tool for the Identification of Thiopeptide Gene Clusters in DNA Sequences

Thiopeptides are a growing class of sulfur-rich, highly modified heterocyclic peptides that are mainly active against Gram-positive bacteria including various drug-resistant pathogens. Recent studies also reveal that many thiopeptides inhibit the proliferation of human cancer cells, further expanding their application potentials for clinical use. Thiopeptide biosynthesis shares a common paradigm, featuring a ribosomally synthesized precursor peptide and conserved posttranslational modifications, to afford a characteristic core system, but differs in tailoring to furnish individual members. Identification of new thiopeptide gene clusters, by taking advantage of increasing information of DNA sequences from bacteria, may facilitate new thiopeptide discovery and enrichment of the unique biosynthetic elements to produce novel drug leads by applying the principle of combinatorial biosynthesis. In this study, we have developed a web-based tool ThioFinder to rapidly identify thiopeptide biosynthetic gene cluster from DNA sequence using a profile Hidden Markov Model approach. Fifty-four new putative thiopeptide biosynthetic gene clusters were found in the sequenced bacterial genomes of previously unknown producing microorganisms. ThioFinder is fully supported by an open-access database ThioBase, which contains the sufficient information of the 99 known thiopeptides regarding the chemical structure, biological activity, producing organism, and biosynthetic gene (cluster) along with the associated genome if available. The ThioFinder website offers researchers a unique resource and great flexibility for sequence analysis of thiopeptide biosynthetic gene clusters. ThioFinder is freely available at http://db-mml.sjtu.edu.cn/ThioFinder/.     

A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.