A Salmonella typhimurium strain defective in uracil catabolism and beta-alanine synthesis

A selection procedure for uracil catabolism mutant strains involving indicator dye plates was developed. Using this method, a strain defective in uracil catabolism has been isolated in Salmonella typhimurium that was temperature-sensitive at 42 degrees C where it required low concentrations of N-carbamoyl-beta-alanine, beta-alanine or pantothenic acid for growth. An extract of the mutant strain degraded uracil at 37 degrees C at a significantly diminished rate compared to that observed for the wild-type strain under the same growth conditions. The conversion of dihydrouracil to N-carbamoyl-beta-alanine was blocked at all temperatures examined in the mutant strain. By means of genetic analysis, the mutant strain was determined to be defective at two genetic loci. Transduction studies with bacteriophage P22 indicated that the panD gene is mutated in this strain, accounting for its beta-alanine requirement. Episomal transfers between Escherichia coli and the mutant strain provided evidence that the defect in uracil catabolism was located in another region of the S. typhimurium chromosome.     

Neuropsychological effects of the CSMD1 genome‐wide associated schizophrenia risk variant rs10503253

The single‐nucleotide polymorphism (SNP) rs10503253, located within the CUB and Sushi multiple domains‐1 (CSMD1) gene on 8p23.2, was recently identified as genome‐wide significant for schizophrenia (SZ), but is of unknown function. We investigated the neurocognitive effects of this CSMD1 variant in vivo in patients and healthy participants using behavioral and imaging measures of brain structure and function. We compared carriers and non‐carriers of the risk ‘A’ allele on measures of neuropsychological performance typically impaired in SZ (general cognitive ability, episodic and working memory and attentional control) in independent samples of Irish patients (n = 387) and controls (n = 171) and German patients (205) and controls (n = 533). Across these groups, the risk ‘A’ allele at CSMD1 was associated with deleterious effects across a number of neurocognitive phenotypes. Specifically, the risk allele was associated with poorer performance on neuropsychological measures of general cognitive ability and memory function but not attentional control. These effects, while significant, were subtle, and varied between samples. Consistent with previous evidence suggesting that CSMD1 may be involved in brain mechanisms related to memory and learning, these data appear to reflect the deleterious effects of the identified ‘A’ risk allele on neurocognitive function, possibly as part of the mechanism by which CSMD1 is associated with SZ risk.     

Diadegma insulare development is altered by Plutella xylostella reared on water‐stressed host plants

Natural enemies of herbivores function in a multitrophic context, and their performance is directly or indirectly influenced by herbivores and their host plants. Very little is known about tritrophic interactions between host plants, pests and their parasitoids, particularly when the host plants are under any stress. Herbivores and their natural enemies’ response to plants under stress are diverse and variable. Therefore, in this study we investigated how diamondback moth, Plutella xylostella (L.), reared on water‐stressed host plants (Brassica napus L. and Sinapis alba L.) influenced the development of its larval parasitoid, Diadegma insulare (Cresson). No significant differences were observed in development of P. xylostella when reared on water‐stressed host plants. However, all results indicated that water stress had a strong effect on developmental parameters of D. insulare. Development of D. insulare was delayed when the parasitoid fed on P. xylostella, reared on stressed host plants. Egg to adult development of D. insulare was faster on non‐stressed B. napus than non‐stressed S. alba followed by stressed B. napus and S. alba. Female parasitoids were heavier on non‐stressed host plants than stressed counterparts. Furthermore, the parasitoid lived significantly longer on stressed B. napus. However, body size was not affected by water treatment. Most host plant parameters measured were significantly lower for water‐stressed than non‐stressed treatments. Results suggest that development of this important and effective P. xylostella parasitoid was influenced by both water stress and host plant species.     

Characterisation of an industrial affinity process used in the manufacturing of digoxin-specific polyclonal Fab fragments

This paper describes the effect of several variables on the affinity process for the production of the FDA approved biotherapeutic product Digoxin Immune Fab (Ovine) (DigiFab, Protherics Inc., TN, USA). The study considers the effects of column re-use on matrix capacity and on the subsequent recovery of the antibody product, and the impact of varying column loading on matrix performance. The methodology used could be equally applied to assess the feasibility of using an affinity matrix for commercial scale purification of alternative antibody derived biotherapeutics. The capacity and specific Fab recovery were calculated through 24h equilibrium and mass balance studies. Results were assessed against data obtained through confocal scanning laser microscopy. Scale-down experiments produced specific Fab recoveries and purities that were comparable with those at production scale. The matrix capacity was found to be 45+/-15 mg of Fab/ml of matrix. Through the use of fluorescent DigiFab and confocal scanning techniques, Fab uptake onto single affinity bead was evaluated. Average intensity values calculated for each sample provided direct real-time, measure of Fab binding and matrix capacity. The results suggest that the affinity matrix had a limited reuse life as a drop in recovery is observed following the completion of a small number of process cycles (30% after three runs). The findings support that which is seen at the current manufacturing scale, where the affinity column is used for a limited number of runs. Results from this study can be used as a basis for future optimisation of this purification process.     

Prion protein fragment PrP-(106-126) induces apoptosis via mitochondrial disruption in human neuronal SH-SY5Y cells

The synthetic peptide PrP-(106-126) has previously been shown to be neurotoxic. Here, for the first time, we report that it induces apoptosis in the human neuroblastoma cell line SH-SY5Y. The earliest detectable apoptotic event in this system is the rapid depolarization of mitochondrial membranes, occurring immediately upon treatment of cells with PrP-(106-126). Subsequent to this, cytochrome c release and caspase activation were observed. Caspase inhibitors demonstrated that while the peptide activates caspases they are not an absolute requirement for apoptosis. Parallel to caspase activation, PrP-(106-126) was also observed to trigger a rise in intracellular calcium through release of mitochondrial calcium stores. This leads to the activation of calpains, another family of proteases. A calpain inhibitor demonstrated that while calpains are activated by the peptide they also are not an absolute requirement for apoptosis. Interestingly a combination of caspase and calpain inhibitors significantly inhibited apoptosis. This illustrates alternative pathways leading to apoptosis via caspases and calpains and that blocking both pathways is required to inhibit apoptosis. These results implicate the mitochondrion as a primary site of action of PrP-(106-126).     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.