Protection of the Lactam Function of 2′-Deoxyinosine with A 2-(4-Nitrophenyl)-Ethyl Moiety

Abstract

The reaction of O-protected inosine with p-nitrophenyl ethanol under Mitsunobu conditions yields a mixture of the 1- and 0° -alkylated derivatives. 2′-Deoxyinosine protected on 0⁶ -, can be synthesized fairly easy from deoxyguanosine with a Mitsunobu reaction followed by reductive deamination.     

Serotonin transporter gene,childhood emotional abuse and cognitive vulnerability to depression

Meta‐analyses evaluating the association between the serotonin transporter polymorphism (5‐HTTLPR) with neuroticism and depression diagnosis as phenotypes have been inconclusive. We examined a gene–environment interaction on a cognitive vulnerability marker of depression, cognitive reactivity (CR) to sad mood. A total of 250 university students of European ancestry were genotyped for the 5‐HTTLPR, including SNP rs25531, a polymorphism of the long allele. Association analysis was performed for neuroticism, CR and depression diagnosis (using a self‐report measure). As an environmental pathogen, self‐reported history of childhood emotional abuse was measured because of its strong relationship with depression. Participants with the homozygous low expressing genotype had high CR if they had experienced childhood emotional maltreatment but low CR if they did not have such experience. This interaction was strongest on the Rumination subscale of the CR measure. The interaction was not significant with neuroticism or depression diagnosis as outcome measures. Our results show that 5‐HTTLPR is related to cognitive vulnerability to depression. Our findings provide evidence for a differential susceptibility genotype rather than a vulnerability genotype, possibly because of the relatively low levels of abuse in our sample. The selection of phenotype and environmental contributor is pivotal in investigating gene–environment interactions in psychiatric disorders.     

Chromomycin, mithramycin, and olivomycin binding sites on heterogeneous deoxyribonucleic acid. Footprinting with (methidiumpropyl-EDTA)iron(II)

The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE x Fe(II)] DNA cleavage inhibition pattern technique. Two DNA restriction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with 32P at the 3' end. MPE x Fe(II) was allowed to partially cleave the restriction fragment preequilibrated with either chromomycin, mithramycin, or olivomycin in the presence of Mg2+. The preferred binding sites for chromomycin, mithramycin, and olivomycin in the presence of Mg2+ appear to be a minimum of 3 base pairs in size containing at least 2 contiguous dG x dC base pairs. Many binding sites are similar for the three antibiotics; chromomycin and olivomycin binding sites are nearly identical. The number of sites protected from MPE x Fe(II) cleavage increases as the concentration of drug is raised. For chromomycin/Mg2+, the preferred sites on the 70 base pairs of DNA examined are (in decreasing affinity) 3'-GGG, CGA greater than CCG, GCC greater than CGA, CCT greater than CTG-5'. The sequence 3'-CGA-5' has different affinities, indicating the importance of either flanking sequences or a nearly bound drug.     

Intra- and interspecific host discrimination in Asobara (Hymenoptera) larval endo-parasitoids of Drosophilidae: Comparison between closely related and less closely related species

Host discrimination studies were conducted with different species of Asobara, which are larval endo-parasitoids of Drosophilidae. Results indicated variable host discrimination which depended on the relatedness of the species. The closely related sibling species Asobara tabida (Nees) and A. rufescens (Foerster) were not only capable of intraspecific discrimination, but also avoided multiparasitism by discriminating between unparasitized host larvae and larvae previously parasitized by females of the other species. This ability to discriminate interspecifically does not seem functional as each species occupies its own microhabitat. As it was shown to be absent in less closely related Asobara species we concluded that interspecific discrimination by A. tabida and A. rufescens was due to their close relationship.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Assay of DNA-RNA hybrids by S1 nuclease digestion and adsorption to DEAE-cellulose filters.

A fast and accurate assay procedure for DNA-RNA hybrids is described in which exhaustive digestion of unhybridized DNA with S1 nuclease is followed by binding of hybrids to filter discs of DEAE-cellulose. The digested DNA can be efficiently washed from the filters so that background levels of 0.1-0.2% of input tracer DNA can be achieved, in contrast to the much higher (approximately 1-5%) backgrounds obtained using TCA precipitation procedures. Short duplexes, as small as 36 nucleotides in length, which are inefficiently bound to hydroxyapatite, are quantitatively bound to the DEAE-cellulose filters.