Cloning of Lentinus edodes mitochondrial DNA fragment capable of autonomous replication in Saccharomyces cerevisiae

Mitochondrial (mt) DNA of the higher basidiomycetes Lentinus edodes with a molecular weight of about 69 kb was partially digested with Sau3AI, cloned with plasmid YIp32 (a hybrid of pBR322 and the yeast leu2 gene) and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. One recombinant plasmid was isolated which contained 3.2 kb fragment of the mtDNA with ARS activity. This plasmid (named pSK52) exhibited a high-frequency yeast transformation and was found to be maintained within the cell as an extrachromosomal element. The stability and copy number properties of pSK52 were similar to those of the recombinant plasmid of YIp32 and S. cerevisiae mt-ARS constructed as a reference. Subcloning experiments were carried out to assess the localization of ARS on the above 3.2 kb fragment, revealing that the fragment contains at least two ARSs.     

Gold complexes with benzimidazole derivatives: synthesis,characterization and biological studies

Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG–DTA) analyses, and FT-IR and UV–Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC₅₀ values below 5.7 μM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells.     

Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S1 nuclease

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S₁ nuclease cleavage. The S₁-cleavage occurred once at these sites. In the absence of added Mg²⁺, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S₁, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S₁-cleavable, unbasepaired sites.     

Senescence Marker Protein 30 Has a Cardio-Protective Role in Doxorubicin-Induced Cardiac Dysfunction

Background

Senescence marker protein 30 (SMP30), which was originally identified as an aging marker protein, is assumed to act as a novel anti-aging factor in the liver, lungs and brain. We hypothesized that SMP30 has cardio-protective function due to its anti-aging and anti-oxidant effects on doxorubicin (DOX)-induced cardiac dysfunction.

Methods and Results

SMP30 knockout (SMP30 KO) mice, SMP30 transgenic (SMP30 TG) mice with cardiac-specific overexpression of SMP30 gene and wild-type (WT) littermate mice at 12–14 weeks of age were given intra-peritoneal injection of DOX (20 mg/kg) or saline. Five days after DOX injection, echocardiography revealed that left ventricular ejection fraction was more severely reduced in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but was preserved in the DOX-treated SMP30 TG mice. Generation of reactive oxygen species and oxidative DNA damage in the myocardium were greater in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but much less in the SMP30 TG mice. The numbers of deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive nuclei in the myocardium, apoptotic signaling pathways such as caspase-3 activity, Bax/Bcl-2 ratio and phosphorylation activity of c-Jun N-terminal kinase were increased in SMP30 KO mice and decreased in SMP30 TG mice compared with WT mice after DOX injection.

Conclusions

SMP30 has a cardio-protective role by anti-oxidative and anti-apoptotic effects in DOX-induced cardiotoxicity, and can be a new therapeutic target to prevent DOX-induced heart failure.     

Cu-metallothioneins (Cu(I)8-MTs) in LEC rat livers 13 weeks after birth still act as antioxidants

Redox properties of metallothioneins (MTs) and Cu in the cytosol from Long-Evans Cinnamon (LEC) rat livers 13 weeks after birth were investigated. MTs from LEC rat livers contain 8 g atoms of Cu and 1 g atom of Zn per mole of protein (Cu(I)8-MTs). Titration of Cu(I)8-MTs with CuCl2 indicates that Cu(I)8-MTs were able to reduce further 2-g atoms of cupric ions per mole MTs as bound form. Hg2+-induced hydroxyl radical generation from Cu(I)8-MTs was demonstrated by ESR using the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The intensity of DMPO-OH signal from Cu-loaded MTs was increased with the increasing number of Cu in MTs. The used cytosol fraction contained 1.37 mM total Cu and 5 mM DTNB titrable-SH groups has a potential to reduce 2 mM CuCl2. No ESR signal due to Cu2+ was also detected with LEC rat liver cytosol, whereas strong Cu2+ signal appeared by the addition of HgCl2. The rate constants for the reaction of Cu(I)8-MTs with superoxide and hydroxyl radicals were estimated to be 2 x 10(6) and > or = 10(12) M(-1)s(-1), respectively, from competition kinetics. Cu2+-catalyzed oxidation of DNA was strongly inhibited both in the presence of Cu-unsaturated MTs and GSH. The results suggest that Cu(I)8-MTs from LEC rat livers just before hepatitis still act as antioxidants.     

D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects

Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.     

Secretory production system of bionanocapsules using a stably transfected insect cell line

Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host–vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 μg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.     

Reaction System of Haemophilus,Superhelical DNA-relaxing Enzyme is Useful for Screening of DNA-binding Substance

Several substances with antioxidant activity were isolated from the mixture of dehydroascorbic acid and tryptophan when reacted together in ethanol. One of the main antioxidant products was obtained in crystalline form from an n-butanol extract of the reaction mixture by means of Sephadex column chromatography followed by HPLC with a reversed phase column. ¹H- and ¹³C-NMR of the product and its acetate showed its structure as a condensate of dehydroascorbic acid and tryptophan with each single molecule involving a C-spiro structure. By a POV test the activity of this substance was about two-thirds of that of BHA on a molar basis, and the activity of the reaction mixture is greatly attributable to this substance.     

Nucleotide sequence homology of the tetracycline-resistance determinant naturally maintained in Bacillus subtilis Marburg 168 chromosome and the tetracycline-resistance gene of B. subtilis plasmid pNS1981

The nucleotide sequence (1579 bp) of tetracycline-resistance determinant and flanking regions of the cloned 5.1 kb DNA fragment from Bacillus subtilis GSY908 chromosome (Sakaguchi, R. and Shishido, K. (1988) Biochim. Biophys. Acta 949, 49-57) were determined and compared with those of the B. subtilis tetracycline-resistance plasmid pNS1981. The tetracycline-resistance structural (tet) genes of the B. subtilis GSY908 chromosome (tetBS908) and pNS1981 (tetpNS1981) were found to be highly homologous (80% identical). Both tet genes were composed of 1374 bp and 458 amino-acid residues initiating from a GTG codon preceded by a ribosome-binding site (RBS-2). Upstream from tetBS908 there exists a short open reading frame (20 amino acids) initiating from a ATG codon preceded by its own RBS (RBS-1). This leader sequence was also highly homologous to that of tetpNS1981 except for a deletion of one bp between the RBS-1 and the ATG codon.