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.     

Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.     

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.     

X-linked recessively inherited non-specific mental retardation. Report of a large family.

A large kindred is described in which 22 males and 3 females show non-specific mental retardation with impaired speech. An X-linked recessive is the most likely mode of inheritance of this condition. Similar families have been described in the literature, characteristic physical abnormalities are absent and performance I.Q. tends to be higher than verbal I.Q. This possible heterogenous condition may be a major individual cause of mental deficiency in males, and may account for the excess of male retardates in the population.