GM risk assessment.

GM risk assessments (GMRAs) play an important role in the decision-making process surrounding the regulation, notification and permission to handle Genetically Modified Organisms (GMOs). Ultimately the role of each GMRA will be able to ensure the safe handling and containment of the GMO; and to asses any potential impacts on the environment and human health. A risk assessment should answer all "what if" scenarios, based on scientific evidence. This article sets out to provide researchers with helpful guidance notes on producing their own GMRA. While reference is made to UK and EU regulations, the underlying principles and points to consider are generic to most countries.

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation.

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5-6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.

Myocarditis as a prognostic indicator in systemic lupus erythematosus.

Lupus myocarditis is a life threatening complication of systemic lupus erythematosus (SLE). A case of left ventricular failure secondary to myocarditis occurring in a patient with SLE is reported. Despite resolution of the cardiac failure with pulsed cyclophosphamide and steroids, she eventually died of non-cardiac complications 18 months later. The literature is also reviewed.

Genetic analysis of in vitro shoot regeneration from cotyledonary petioles of Brassica oleracea.

Diallel analysis was used to investigate the genetic control of in vitro shoot regeneration in Brassica oleracea. Twelve doubled haploid (DH) lines, selected to include a range of genotypes with differing shoot regeneration potentials, were crossed reciprocally to produce 132 F(1) and 12 selfed, DH families. Cotyledonary petioles from 4-day-old seedlings, from all families, were excised and maintained on MS medium supplemented with 2 mg/l BAP. Explants were scored after 44 days for both the presence or absence of shoots and the number of regenerating shoots per explant. Diallel analysis showed both shoot regeneration and the production of multiple shoots to be controlled by additive and dominant gene effects, with additive effects being more important. Additive gene effects accounted for 71% and 77% of the genetic variation observed within the diallel for shoot regeneration and multiple shoot regeneration, respectively. By investigating the shoot regeneration potential of subsequent backcross and F(2) populations, the ability to introduce and increase shoot regeneration potential into otherwise recalcitrant lines was demonstrated.

Genetic analysis of Agrobacterium tumefaciens susceptibility in Brassica oleracea.

The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map. Preliminary studies were carried out by analysis of an 8 x 8 diallel, for which the parental lines were selected to include a range of susceptibilities to A. tumefaciens. The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important. A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects. A high narrow-sense heritability value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high. Fifty-nine DH lines from the mapping population were screened for susceptibility towards A. tumefaciens. Variation in susceptibility was observed across the population. The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A. tumefaciens was identified on linkage group 09. The use of substitution lines covering this region confirmed the location of this QTL. This work shows that susceptibility to A. tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated. These findings may help to overcome genotype restrictions to genetic transformation.

Influence of dose range on degree of nonlinearity detected in dose-proportionality studies for drugs with saturable elimination: single-dose and steady-state studies.

Deviation from proportionality occurs when the ratio of area under the curve (AUC) values is not equal to the ratio of administered doses. The degree of nonlinearity (fNL) can be quantitated as the ratio of AUCs divided by the ratio of doses. We explore positive deviation from proportionality (fNL > 1) using the classical Michaelis-Menten model of nonlinear elimination after a single dose (n = 1) or at steady state (ss). The degree of nonlinearity is related to the ratio of the highest dose to the lowest dose (Rd = DH/DL): fn = 1NL = (2 + Rd.epsilon)/(2 + epsilon), fssNL = (Rd.omega - 1)/(Rd.omega - Rd), where epsilon is the ratio of the initial concentration after the lowest dose to the Km (epsilon = DL/Km.V) and omega is the ratio of the Vmax to the average rate of input for the highest dose (omega = Vmax tau/F.DH). From these relationships, we find that (1) for single-dose studies, Km is the important Michaelis-Menten parameter, while Vmax is important at steady state; (2) the degree of nonlinearity cannot exceed the ratio of doses in single-dose studies, and when doses in extreme excess of Km.V are chosen, the degree of nonlinearity is equal to the dose range; and (3) at steady state, the degree of nonlinearity can exceed the ratio of doses and approaches infinity as the average input rate approaches Vmax. Literature data (phenytoin and ethanol) support these findings. We conclude that the degree of nonlinearity is not a useful measure of nonlinearity in and of itself and propose percentage saturation as being more informative.

Studies with two investigational interventions: a simple model to assess their effects and interaction.

The paper presents a simple way to design and analyse studies on pharmacodynamic food-interaction and drug-interaction studies. The study design includes two distinct interventions--the drug or control against eating, or fasting, and allows an analysis of variance. It provides a model for a more analytical approach in the conduct and analysis of pharmacodynamic interaction studies and pharmacotherapeutic combination trials.

Effects of age, tenure, training, and job complexity on technical performance.

Effects on performance of age, tenure, training level, and job complexity were investigated in a cross-sectional study using a sample of 1,308 service engineers employed by a multinational office equipment company. Two measures of job performance were derived from production record data, one relating to the quality of servicing and the other to the speed with which services were completed. Scores for each performance measure were analyzed by analysis of variance. For the quality of servicing measure, a significant main effect of age and a significant Age X Training interaction were obtained, and the relation between age and job performance took the form of an inverted U. For the speed of servicing measure, the main effects of age, tenure, training level, and job complexity were significant and there were no significant interactions. However, for both performance measures, age accounted for only a very small proportion of the variance. We discuss these results with reference to the existing literature on age and technical job performance, and conclude that training, especially if it is recent, may moderate adverse effects of age on job performance.

Purification and characterisation of ovine C4: evidence for two molecular forms in ovine plasma.

A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the alpha chain--108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated alpha' chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the alpha and beta chains were lowered following digestion of ovine C4 with neuraminidase.

Purification and characterisation of the fourth component of bovine complement.

A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.

Electrocardiographic evaluation of the healthy raccoon (Procyon lotor).

Leads I, aVF, V3, and V10 ECG were obtained from 12 healthy raccoons anesthetized with xylazine and ketamine. Intervals and amplitudes of component deflections were remarkably uniform among the individuals. Vector orientation of QRS mimicked that for the dog; but the relatively low voltages to QRS mimicked those of cats. The QT interval varied inversely with heart rate, whereas the PQ interval varied directly with body weight. Relationship between heart rate and body weight was not significant (P greater than 0.05).