Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.     

Racial differences in women's desires to be thin

The goal of this research was to attempt to understand why white women are more prone to develop eating disorders than black women. Using self-reports, we found that white women chose a significantly thinner ideal body size than did black women, and expressed more concern than black women with weight and dieting. White women also experienced greater social pressure to be thin than did black women. White men indicated less desire than black men to date a women with a heavier than ideal body size, and white men felt they would more likely be ridiculed than did black men if they did date a woman who was larger than the ideal. The results suggest that black women experience eating disorders less than white women at least in part because they experience less pressure to be thin.     

Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apol/CD95. One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.     

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse--I. Naturally occurring variability in active zone structure

The orderly arrays of intramembranous particles (IMPs) found in the p-face of freeze-fracture replicas of the frog neuromuscular junction ('active zones') are believed to be involved in transmitter release. Some or all of the particles represent voltage-dependent Ca2+ channels. Since there is a great heterogeneity in the amount of transmitter released by different frog motor nerve terminals we sought to determine whether active zone (AZ) structure displayed a similar heterogeneity by using a novel freeze-fracture procedure providing large, intact replicas containing significant portions of motor nerve terminals from the cutaneous pectoris muscle of the frog, Rana pipiens. Using only junctions in which more than 50 AZs or more than 50 microm of nerve terminal were included in the fractures, we measured AZ length, AZ intramembranous particle density, terminal width at each AZ, space between AZs, the angle of AZ orientation with respect to the longitudinal axis of the nerve terminal, exposed pre-synaptic nerve terminal surface area and a calculated value for mean AZ length per unit terminal length. The analysis led to the following conclusions. There is an approximate 5-fold range in mean AZ length/micrometre terminal length. Terminal width is a good predictor of AZ length. Particle density does not vary significantly within a given AZ, nor between AZs from the same or different junctions. The distance between AZs is not related to AZ length, i.e. shorter AZs are no more or less likely to be closer to the adjacent AZ. The probability of release from any AZ on action potential invasion is small. If most of the IMPs are Ca2+ channels, either the probability of channel opening or the efficacy of triggering release is very low or both. That the variability in release efficacy in different terminals is much greater than ultrastructural variability in terminals suggests that regulation of release is dominated by physiological processes that do not have obvious ultrastructural correlates. On the other hand, the apparent excess of AZ relative to the number of vesicles released indicates that the amount and variability in amount of AZ is important in ways that need to be elucidated.     

The activity of the high-affinity K+ uptake system Kdp sensitizes cells of Escherichia coli to methylglyoxal

Expression of the Kdp system sensitizes cells to methylglyoxal (MG) whether this electrophile is added externally or is synthesized endogenously. The basis of this enhanced sensitivity is the maintenance of a higher cytoplasmic pH (pHi) in cells expressing Kdp. In such cells, MG elicits rapid cytoplasmic acidification via KefB and KefC, but the steady-state pHi attained is still too high to confer protection Lowering pHi further by incubation with acetate increases the sensitivity of cells to MG.     

Human influenza virus A/HongKong/156/97 (H5N1) infection

Introduction of influenza viruses with gene segments of avian origin into the human population may result in the emergence of new pathogenic human influenza viruses. The recent infection of a 3-year-old boy with an influenza A (H5N1) virus of avian origin can be considered as an example of such an event. However, this virus, influenza A/Hong Kong/156/97 (H5N1) and the 17 additional H5N1 viruses isolated from humans by the end of 1997 lack the ability to spread efficiently amongst humans and therefore have limited pandemic potential. However, the possibility of reassortment of these viruses with currently circulating human viruses illustrates the need for pandemic preparedness.