PCR and magnetic bead-mediated target capture for the isolation of short interspersed nucleotide elements in fishes

Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180-360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNA(Leu)-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs.     

硅烷Y9669改性湿固化聚氨酯热熔胶的研制

以4,4′-二苯基甲烷二异氰酸酯(MDI)、聚醚多元醇(N-210)和聚己二酸己二醇酯(PHA)为主要原料,控制R=n(-NCO)/n(-OH)=2.0,制得PUR(湿固化聚氨酯热熔胶)的预聚体;然后以不同的硅烷偶联剂[如Y9669(N-苯基-γ-氨丙基三甲氧基硅烷)、KH-550和KH-560等]作为PUR中部分端-NCO基的封端剂,制备SPUR(硅烷化PUR)。结果表明:Y9669是较理想的封端剂,能使SPUR的剪切强度增加37.23%;SPUR的硬度、热稳定性能随Y9669封端率增加而增大;SPUR的剪切强度随Y9669封端率增加呈先升后降态势,并且在Y9669封端率为20%时相对最大(17.73 MPa)。     

Investigations on the role of acyl initiators and CO2 in nylon 4 polymerization

Summary The preparation of high molecular weight nylon 4 with alkali metal pyrrolidonate catalysts, acyl initiators, and CO₂ was studied. Isolated sodium pyrrolidonate and the appropriate amount of acyl initiator gave unusually high molecular weight nylon 4 (m_w >200,000). The molecular weight-initiator concentration relationship indicates that acyl compounds play a dual role in the polymerization, acting both as initiators and scavengers for chain-terminating impurities. One potential chain terminator was identified as 4-aminobutyric acid. The high polymer molecular weights obtained in the presence of CO₂ can also be explained by the scavenging of chain terminators by CO₂. Determination of the number of growing chains, however, shows that CO₂, unlike the acyl compounds, is not an initiator in nylon 4 polymerization.Presented at the 1984 International Congress of Pacific Basin Societies, Honolulu, Hawaii, December 1984     

Human Recombinant DNase I (Pulmozyme®) Inhibits Lung Metastases in Murine Metastatic B16 Melanoma Model That Correlates with Restoration of the DNase Activity and the Decrease SINE/LINE and c-Myc Fragments in Blood Cell-Free DNA

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme^®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme^® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme^® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme^®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.     

Methylene blue as a retarder of free radical polymerization: 1. Polymerization of acrylonitrile,methyl methacrylate and styrene

Methylene blue (MB), a redox dye, in monomeric form significantly retards the free radical polymerization of styrene (ST) and acrylonitrile (AN) in N,N-dimethyl formamide solvent. The effect is small in methyl methacrylate (MMA) polymerization. Partly reduced MB is found to be a stronger retarder than MB. The rate constants for the oxidation of the polymer radicals by MB at 60°C follows the order ST>AN>MMA. The results cannot be explained when only taking into consideration the reactivity and polarity parameters of the radicals and the substrate, as is done in the Q-e scheme. The low rate constant with the PMMA radical is attributed to steric effects. The current work has shown that single molecules of MB are effective chain terminators and this is in sharp contrast with the conclusion reached by Chen who postulated that only the aggregates of MB are the chain terminators in the polymerization of acrylamide in water.     

Effects of Polymorphisms in Pepsinogen (PEP), Amylase (AMY) and Trypsin (TRY) Genes on Food Habit Domestication Traits in Mandarin Fish

Mandarin fish (Siniperca chuatsi) have a peculiar feeding habit of only accepting live fish prey and refusing dead prey and artificial diets. However, previous research has shown that some individuals accept dead prey after gradual domestication. Digestive enzymes are correlated with feeding habits in fish. In the current study, SNPs in the mandarin fish genes for pepsinogen (PEP), amylase (AMY), and trypsin (TRY) were evaluated for associations with feeding habits in domesticated mandarin fish by scanning their complete genomic sequence. In total, two SNPs were found in PEP, one was found in TRY, and none were found in AMY. The D1(CTCC) and D5(TTTT) diplotypes in the PEP gene tended to show strong effects on the feeding habits of domesticated fish (p < 0.01). The results indicate that PEP may be associated with the genetic mechanism for feeding habits in mandarin fish, and the D1(CTCC) and D5(TTTT) diplotypes in the PEP gene may be useful markers for selecting mandarin fish with appropriate feeding habits for domestication.     

Adsorption of rhodium(III) ions onto poly(1,8-diaminonaphthalene) chelating polymer: Equilibrium,kinetic and thermodynamic study

In the present work, poly(1,8-diaminonaphthalene) (poly(1,8-DAN)) was synthesized by the reaction of 1,8-diaminonaphthalene (1,8-DAN) with ammonium persulfate (APS) and then the equilibrium, kinetics and thermodynamics of rhodium(III) adsorption onto poly(1,8-DAN) were studied. Poly(1,8-DAN), Rh(III)-poly(1,8-DAN) and Rh(III)-1,8-DAN complex were characterized by UV–vis. and FTIR spectroscopy, thermal analysis, potentiometric titration and electrical conductivity. In the adsorption studies, the effects of acidity, the temperature and the concentration of rhodium(III) were examined. It was found that poly(1,8-DAN) has Rh(III) adsorption capacity (q_m) of 11.11 mg/g polymer. The adsorption data fitted better to the Freundlich isotherm then the Langmuir isotherm, and the kinetics of the adsorption fitted to pseudo second order kinetic model. The ΔG° values were calculated as ?7.33 at 20 and ?11.31 kJ/mol at 60 °C. The enthalpy (ΔH°), entropy (ΔS°) and the activation energy (E_a) of the adsorption were found as 21.335 kJ/mol, 97.057 J/mol K and 70.210 kJ/mol, respectively. It was predicted that the adsorption of Rh(III) onto poly(1,8-DAN) was an endothermic chemical adsorption process governed by both ionic interaction and chelating mechanisms. It was also observed that the adsorption of Rh(III) lowered the electrical conductivity of the pol(1,8-DAN).     

Identification and Characterization of Buffalo 7SK and U6 pol III Promoters and Application for Expression of Short Hairpin RNAs

RNA polymerase III (pol III) type 3 promoters, such as 7SK and U6, are routinely used to induce short hairpin RNAs (shRNAs) to knockdown gene expression by RNA interference (RNAi). To extend the application of RNAi to studies of buffalo, an shRNAs expressing system using the buffalo pol III promoters was developed. Buffalo 7SK promoter (bu7SK) and U6 promoter (buU6) sequences upstream of the full-length 7SK and U6 small nuclear RNA sequence in the buffalo genome were identified and characterized, respectively. To determine the functionality of these promoters in constructs driving shRNA expression, anti-EGFP shRNAs (shEGFP) cassettes under the direction of bu7SK and buU6 were constructed. We further compared the EGFP knockdown efficiency of constructs using bu7SK and buU6 with that of promoters of human and bovine origins in BFF cells and mouse PT67 cells by flow cytometry and quantitative real-time PCR assays. We found that the bu7SK and buU6 promoters induced the greatest level of suppression in homologous and heterologous cells relative to promoters derived from other species. Taken together, functional bu7SK and buU6 promoters were identified and characterized, thus laying the groundwork for future development of RNAi therapeutics and gene modification in buffalo species.