Parallel-Stranded Duplex DNA and Self-Assembled Quartet Structures Formed by Isoguanine and Related Bases

Abstract

Parallel-stranded (ps) oligonucleotide duplexes are described containing isoguanine-cytosine and/or 5-methylisocytosine-guanine base pairs. A parallel hybrid is also formed when 5-aza-7-deazaguanine base pairs with guanine while the base pair with isoguanine results in an antiparallel duplex. Oligomers such as d(T₄isoG₄T₄) form selfassembled tetraplexes which show a cation selectivity different from that of the G-quartet.     

Four-Stranded Intercalated Cytosine-Rich Molecules: Novel Insights into DNA Structure and Stability

Abstract

DNA fragments with stretches of cytosine residues can form four-stranded intercalated i-DNA molecules stabilized by hemiprotonated cytosine·cytosine⁺ (C·C⁺) base pairs. Intriguing features of this motif are the accomodation of base stacking that is unfavorable due to electrostatic repulsion and the close approach of phosphates in narrow grooves of the molecule. Unusual sources of stability in this structure involve sugar-base stacking and CH-O interribose short contacts between the backbones of adjacent strands.     

Thermal Characterization and Cytotoxicity of Complexes Formed by Papain and Cyclodextrin

Papain is a proteolytic enzyme with restricted applications due to its limited stability. Cyclodextrins are widely used in pharmaceutical formulations once they are able to form complexes with other molecules, improving their stability and bioavailability. The purpose of the present paper was to analyze complexes formed by papain/hydroxypropyl-β-cyclodextrin and papain/β-cyclodextrin by thermal analysis and cytotoxicity tests to verify their possible interactions and toxicological behavior. Complex solutions were prepared at different molar ratios and collected as a function of time to be lyophilized and analyzed. Results showed changes in endothermic events and cytotoxicity profiles. A complex formation for both complexes is observed; nevertheless, β-cyclodextrin was able to change the enzyme characteristics more efficiently.     

Strategies for Pyramiding Resistance Genes Against the Barley Yellow Mosaic Virus Complex (BaMMV,BaYMV, BaYMV-2)

Barley Yellow Mosaic Virus disease caused by different strains of BaYMV and BaMMV is a major threat to winter barley cultivation in Europe. Pyramiding of resistance genes may be considered as a promising strategy to avoid the selection of new virus strains and to create more durable resistances. However, this goal cannot be achieved by phenotypic selection due to the lack of differentiating virus strains. For pyramiding of resistance genes rym4, rym5, rym9 and rym11, located on chromosomes 3H and 4H of barley two different strategies have been developed. These strategies are based on doubled haploid lines (DHs) and marker assisted selection procedures. On the one hand F₁ derived DH-plants of single crosses were screened by molecular markers for genotypes being homozygous recessive for both resistance genes. These genotypes were crossed to lines carrying one resistance gene in common and an additional third gene, leading to a DH-population of which 25% carry three resistance genes, 50% have two resistance genes and 25% possess a single resistance gene homozygous recessively. Alternatively, F₁ plants having one resistance gene in common were directly inter-crossed [e.g. (rym4 × rym9) × (rym4 × rym11)] and about 100 seeds were produced per combination. Within these complex cross progenies plants were identified by markers being homozygous at the common resistance locus and heterozygous at the others. From such plants, theoretically present at a frequency of 6.25%, DH-lines were produced, which were screened for the presence of genotypes carrying three or two recessive resistance genes in a homozygous state. Besides DH-plants carrying all possible two-gene combinations, 20 DH-plants out of 107 analysed carrying rym4, rym9, and rym11 and 27 out of 187 tested carrying rym5, rym9, and rym11 homozygously have been detected using the second strategy which is faster but needs co-dominant markers, because in contrast to the first strategy marker selection is carried out on heterozygous genotypes.     

Structural Study of the Complex Formed by Ceruloplasmin and Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a key proinflammatory cytokine. Inhibitors of tautomerase activity of MIF are perspective antiinflammatory compounds. Ceruloplasmin, the copper-containing ferroxidase of blood plasma, is a noncompetitive inhibitor of tautomerase activity of MIF in the reaction with p-hydroxyphenylpyruvate. Small-angle X-ray scattering established a model of the complex formed by MIF and ceruloplasmin. Crystallographic analysis of MIF with a modified active site supports the model. The stoichiometry of 3 CP/MIF trimer complex was established using gel filtration. Conformity of novel data concerning the interaction regions in the studied proteins with previous biochemical data is discussed.     

Supplementary Chromium(III) Propionate Complex Does Not Protect Against Insulin Resistance in High-Fat-Fed Rats

Improper eating habits such as high-fat or high-carbohydrate diets are responsible for metabolic changes resulting in impaired glucose tolerance, hyperinsulinemia, insulin resistance, and ultimately diabetes. Although the essentiality of trivalent chromium for humans has been recently questioned by researchers, pharmacological dosages of this element can improve insulin sensitivity in experimental animals and diabetic subjects. The aim of the study was to assess the preventive potential of the supplementary chromium(III) propionate complex (CrProp) in rats fed a high-fat diet. The experiment was conducted on 32 male Wistar rats divided into four groups and fed the following diets: the control (C, AIN-93G), high-fat diets (HF, 40 % energy from fat), and a high-fat diet supplemented with CrProp at dosages of 10 and 50 mg Cr/kg diet (HF?+?Cr10 and HF?+?Cr50, respectively). After 8 weeks, high-fat feeding led to an increased body mass, hyperinsulinemia, insulin resistance, a decreased serum urea concentration, accumulation of lipid droplets in hepatocytes, and increased renal Fe and splenic Cu contents. Supplementary CrProp in both dosages did not alleviate these changes but increased renal Cr content and normalized splenic Cu content in high-fat-fed rats. Supplementary CrProp does not prevent the development of insulin resistance in rats fed a high-fat diet.     

Storage Stability and Thermal Stability of Hordeumin,an Anthocyanin Pigment from Barley

Hordeumin stored at –40 to –80^oC in 1% HCI–methanol suffered neither from color reduction nor discoloration. After heating at 80°C for 60 min, hordeumin showed a pigment retention rate of 100%. This characteristic is because the pigment is a composite high-molecular weight compound consisting of anthocyanins and polyphenols, It was determined, however, that discoloration and browning occurred more rapidly than color reduction during storage and heating of the pigment.     

Isoguanine quartets formed by d(T4isoG4T4): tetraplex identification and stability.

The self-aggregation of the oligonucleotide d(T4isoG4T4) (1) is investigated. Based on ion exchange HPLC experiments and CD spectroscopy, a tetrameric structure is identified. This structure was formed in the presence of sodium ions and shows almost the same chromatographic mobility on ion exchange HPLC as d(T4G4T4) (2). The ratio of aggregate versus monomer is temperature dependent and the tetraplex of [d(T4isoG4T4)]4 is more stable than that of [d(T4G4T4)]4. A mixture of d(T4isoG4T4) and d(T4G4T4) forms mixed tetraplexes containing strands of d(T4isoG4T4) and d(T4G4T4).     

Acetylation of Dna2 Endonuclease/Helicase and Flap Endonuclease 1 by p300 Promotes DNA Stability by Creating Long Flap Intermediates

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α.     

Ubiquitin-dependent and Ubiquitin-independent Control of Subunit Stoichiometry in the SWI/SNF Complex

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.     

复合诱变和抗性筛选利迪链菌素高产菌株

为解决利迪链霉菌生产能力低的问题,以Streptomyces lydicus AS4.2501-P28为出发菌株,应用紫外和吖啶橙诱变,结合抗生素的抗性筛选,得到稳定高产的S.lydicus AS4.2501-L8,发酵水平达到177.0μg/mL,较出发菌株提高了96.2%。     

HearNPV Pseudotyped with PIF1, 2, and 3 from MabrNPV: Infectivity and Complex Stability

Effective oral infection is set off by interaction of a group of conserved per os infectivity factors (PIFs) with larval midgut columnar epithelial cells. We constructed pseudotyped viruses by substituting pif1, pif2 or pif3 genes of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) with their homologs from Mamestra bracissae multiple nucleopolyhedrovirus and tested their infectivity to tissue culture cells and to larvae. Transfection and infection assays revealed that all recombinant viruses generated infectious budded virus in both cell culture and in larvae. Electron microscopy showed synthesized occlusion body and occlusion derived virus (ODV) were morphologically indistinguishable from those of the parental virus. By contrast, feeding assays revealed that pseudotyped viruses could not rescue oral infectivity except for pif3 pseudotyped virus that only partially rescued oral infectivity but at a mortality rate much lower than that of the parental HearNPV. Consistent with the bioassay result, PIF complex was detected in ODVs of pif3 pseudotyped virus only but not in pif1 or pif2 pseudotyped viruses. Our results suggest that PIF complex is essential for oral infectivity, and in the formation of the PIF complex, PIF1, 2 are virus-specific while PIF3 does not appear to be as specific and can function in heterologous environment, albeit to a much more limited extent.     

复合诱变和抗性筛选利迪链菌素高产菌株

为解决利迪链霉菌生产能力低的问题,以Streptomyces lydicus AS 4.2501-P28为出发菌株,应用紫外和吖啶橙诱变,结合抗生素的抗性筛选,得到稳定高产的S. lydicus AS 4.2501-L8,发酵水平达到177.0 μg/mL,较出发菌株提高了96.2%。     

Plant Resistance Suppressors in the Pathosystem Formed by Potato and the Causal Agent of Late Blight

The properties and effects of two plant resistance suppressors (1,3--1,6--glucan and a pentasaccharide of xyloglucan origin) involved in the pathosystem of potato (Solanum tuberosum) and the causal agent of blight (Phytophthora infestans(Mont) de Bary) were compared. The microbial 1,3--1,6--glucan suppressed the defense response over a narrow concentration range (10^–2M), whereas the plant pentasaccharide had a broad range of effective concentrations (10^–12to 10^–6M). In the pathosystem of potato and the causal agent of late blight, the -glucan caused a local and race-specific suppressor effect on the plant host defense response. In contrast, the pentasaccharide caused both local and systemic suppression of potato resistance and the presence of terminal fucosyl residue in the xyloglucan oligosaccharine played a decisive role in its effect. The recognition of both suppressors by potato cell membrane sites is discussed.     

Spore production of Penicillium roqueforti by solid state fermentation: Stoichiometry, growth and sporulation behavior

The development of Penicillium roqueforti on buckwheat seeds proceeds roughly into four steps, involving a lag phase and three growth phases. First, it appears as a spore germination and external colonization of the grains by the mycelium. Then, mainly external sporulation and internal colonization of the seeds occur and finally internal sporulation takes place. The Stoichiometry of the growth and the sporulation is established. Kinetic experiments performed in a fixed bed reactor show that the growth of the microorganism (biomass production) may be estimated by the protein content of the medium. This growth occurs with a very low mu(max) value close to 0.030 h(-1). The chitin content of the medium is an indicator of the sporulation, just as the metabolic liquor (mainly water) produced during the course of a cultivation. The values of the observed respiratory quotient are close to those predicted by stoichiometry.     

Enhanced Thermal Stability and Biocompatibility of Gold Nanorods by Graphene Oxide

In the present study, the effect of nanosized graphene oxide layer on thermal stability and biocompatibility of gold nanorods has been examined. The graphene oxide-wrapped gold nanorods were prepared by electrostatic interaction between negatively charged graphene oxide and positively charged nanorods. The resulting nanohybrids were then heated at different time intervals to 95 °C in a water bath to assess the effect of heat on the rods morphology. The structural changes in gold nanorods were monitored via UV-Vis spectroscopy measurements and transmission electron microscopy images. In similar experiments, the graphene oxide used to wrap gold nanorods was reduced by ascorbic acid in a 95 °C water bath. Our results indicate that while bare gold nanorods are highly vulnerable to elevated temperatures, graphene oxide and reduced graphene oxide-coated gold nanorods remain thermally stable with no structural changes. We also confirmed that the enhanced thermal stability is highly dependent on the concentration of deposited graphene oxide available on the surface of the gold nanorods. In addition, we performed an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazoliumbromide) assay to make a comparison between the cytotoxicity of the nanohybrids and their primary building blocks on human dermal fibroblast cells as a normal cell line. We found evidence that graphene oxide can enhance the biocompatibility of the rods through covering toxic chemicals on the surface of them.

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Graphical Abstract ?

     

鸡新城疫口服DNA疫苗的安全性、稳定性与免疫效力

将含新城疫病毒（NDV）F₄₈E₉株融合蛋白（F）基因的真核表达质粒pcDNA3-F的减毒鼠伤寒沙门氏菌ZJ111株（ZJ111/pcDAN3-F）口服接种小鼠和雏鸡。结果表明：利用该减毒株作为载体传递DNA疫苗具有相对安全性。用酶切和PCR鉴定法证实在体内外无抗生素存在的条件下重组质粒在受体菌ZJ111菌株内比较稳定。ZJ111/pcDAN3-F以每羽10⁸cfu免疫雏鸡,2周后二免,二免后4周攻击致死剂量的强毒株F₄₈E₉,观察其免疫效力。结果表明：重组ZJ111/pcDNA3-F菌株不仅能诱导雏鸡产生抗NDV抗体,而且能诱导法氏囊B淋巴细胞和胸腺T淋巴细胞的增殖反应,对强毒株攻击的保护率为66.7%,高于pcDNA3-F裸质粒DNA疫苗注射免疫组（50%）。上述结果初步提示减毒沙门氏菌为载体传递DNA疫苗具有良好的安全性、稳定性和免疫原性,为研制低成本、实用化的口服禽类DNA疫苗奠定了基础。