Cellular assay optimization: part I: the use of large-scale transiently transfected cryobanks and introduction of a c-Myc tag to design a standardized ELISA process

This study investigated the use of large-scale transiently transfected cryopreserved cells for medium-throughput cellular screening. The data generated indicated that preprepared transiently transfected cryobanks can be used for cell-based assays and in fact can greatly enhance the consistency of data generated by cellular screens. In addition to this, a generic enzyme-linked immunosorbent assay method was designed that introduced a c-Myc tag to four different targets and allowed all four cell assays to be run using a standardized process. These process improvements yielded cost savings and greatly reduced the required resource, as well as reducing timelines for developing cellular assays.     

Crayfish recognize the faces of fight opponents

The capacity to associate stimuli underlies many cognitive abilities, including recognition, in humans and other animals. Vertebrates process different categories of information separately and then reassemble the distilled information for unique identification, storage and recall. Invertebrates have fewer neural networks and fewer neural processing options so study of their behavior may reveal underlying mechanisms still not fully understood for any animal. Some invertebrates form complex social colonies and are capable of visual memory-bees and wasps, for example. This ability would not be predicted in species that interact in random pairs without strong social cohesion; for example, crayfish. They have chemical memory but the extent to which they remember visual features is unknown. Here we demonstrate that the crayfish Cherax destructor is capable of visual recognition of individuals. The simplicity of their interactions allowed us to examine the behavior and some characteristics of the visual features involved. We showed that facial features are learned during face-to-face fights, that highly variable cues are used, that the type of variability is important, and that the learning is context-dependent. We also tested whether it is possible to engineer false identifications and for animals to distinguish between twin opponents.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.     

Effects of low concentrations of cetyltrimethylammonium bromide on diluted bovine spermatozoa

Cetyltrimethylammonium bromide (CTAB) at a concentration of 10 mg/l in a citrate buffer partially protected sperm against the effects of cold shock treatment. CTAB concentrations of 10 and 20 mg/l also increased in vitro sperm livability when semen was diluted in Caprogen and incubated at 37 degrees C (50 h v. 60 and 57 h). Concentrations of CTAB exceeding 50 mg/l reduced in vitro sperm livability. The addition of catalase (4.5 mg/l) to Caprogen produced greater increases in in vitro sperm livability (50 h v. 110 h), but did not eliminate the toxic effect of the higher CTAB concentrations. The addition of CTAB (10 mg/l) to the Caprogen-catalase diluent significantly increased the non-return rate of the diluted semen in only one of four large-scale field trials and did not alter non-return rates with deep-frozen semen.     

d'plus: A program to calculate accuracy and bias measures from detection and discrimination data

The program d'plus calculates accuracy (sensitivity) and response-bias parameters using Signal Detection Theory. Choice Theory, and 'nonparametric' models. is is appropriate for data from one-interval, two- and three-interval forced-choice, same different, ABX, and oddity experimental paradigms.