The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

Changes in performance,maximal oxygen uptake and maximal accumulated oxygen deficit after 5, 10 and 15 days of live high:train low altitude exposure

Nineteen well-trained cyclists (14 males and 5 females, mean initial V˙O_2max 62.3 ml kg^–1 min^–1) completed a multistage cycle ergometer test to determine maximal mean power output in 4 min (MMPO_4min), maximal oxygen uptake (V˙O_2max) and maximal accumulated oxygen deficit (MAOD). The athletes were divided into three groups, each of which completed 5, 10 or 15 days of both a control condition (C) and live high:train low altitude exposure (LHTL). The C groups lived and trained at the ambient altitude of 610 m. The LHTL groups spent 8–10 h night^–1 in normobaric hypoxia at a simulated altitude of 2,650 m, and trained at the ambient altitude of 610 m. The changes to MMPO_4min, V˙O_2max and MAOD in response to LHTL altitude exposure were not significantly different for the 5-, 10- and 15-day treatment periods. For the pooled data from all three treatment periods, there were significant increases in MMPO_4min [mean (SD) 5.15 (0.83) W kg^–1 vs 5.34 (0.78) W kg^–1] and MAOD [50.1 (14.2) ml kg^–1 vs 54.9 (13.1) ml kg^–1] in the LHTL athletes between pre- and post-altitude exposure. There were no significant changes in MMPO_4min [5.09 (0.76) W kg^–1 vs 5.16 (0.86) W kg^–1] or MAOD [50.5 (14.1) ml kg^–1 vs 49.1 (13.0) ml kg^–1] in the C athletes over the corresponding period. There were significant increases in V˙O_2max in the athletes during both the LHTL [63.2 (9.0) ml kg^–1 min^–1 vs 64.1 (9.0) ml kg^–1 min^–1] and C [62.0 (8.6) ml kg^–1 min^–1 vs 63.4 (9.2) ml kg^–1 min^–1] conditions. In these athletes, there was no difference in the impact of 5, 10 or 15 days of LHTL on the increases observed in MMPO_4min, V˙O_2max or MAOD; and LHTL increased MMPO_4min and MAOD more than training at low altitude alone. Electronic Publication     

A model for susceptibility artefacts from respiration in functional echo-planar magnetic resonance imaging

Respiration causes variations in the signals acquired during magnetic resonance imaging (MRI) and therefore is a significant source of noise in functional brain imaging. A primary component of respiratory noise may arise from variations of bulk susceptibility or air volume in the chest. Here we investigate the nature of the image artefacts that can be caused by such changes. We develop a simple model which attempts to mimic the effects of variations in susceptibility and volume during respiration. Theoretical calculations, computer simulations and imaging experiments with this model show that small variations in susceptibility within the thorax from alterations in the paramagnetism of cavity gas may lead to a shift of the image on the order of 0.1 pixels as well as a shading of the intensity by +/-1%. These effects are observed to be predominant in the phase-encoding direction. They may lead to the production of spurious activations in functional MRI and are likely to be of more importance at higher field strengths.