Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate

One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. (32)P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo(2)-[4'-(32)P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.     

Can salinity-induced mortality explain larval vertical distribution with respect to a halocline?

For the larvae of two echinoderm species that coexist in Atlantic Canada (bipinnaria of the sea star Asterias rubens and 4- and 6-arm echinoplutei of the sea urchin Strongylocentrotus droebachiensis), we examined the effect of short- and long-term exposure to salinity (ranging from 18 to 35) on the probability of larval survival in laboratory experiments. We also related larval vertical distributions in response to sharp haloclines generated in the laboratory to survival probability in the salinity of different layers in the water column. For both species and developmental stages, survival probability decreased with decreasing salinity, and a salinity range of 24-27 emerged as the critical threshold for larval tolerance. The relationship between the proportion of larvae that crossed a halocline into the top water layer and the survival probability of larvae in the salinity of that layer was significant for both species. Interestingly, the shape of this response was species-specific but not stage-specific for S. droebachiensis. Our findings suggest that larval avoidance of low-salinity water layers may be an adaptive behavior that increases survival and indirectly influences larval distribution.     

Estrogen, medroxyprogesterone acetate, endothelial function, and biomarkers of cardiovascular risk in young women

Medroxyprogesterone acetate (MPA) is widely known for its use in combination hormone therapy for postmenopausal women. However, MPA is also commonly used in young women for contraception and treatment of a number of gynecological conditions. Despite its widespread use, the cardiovascular effects of MPA in young women are unclear. Therefore, the purpose of this study was to determine the acute effects of MPA when used in combination with estradiol on markers of cardiovascular risk in young women. We suppressed endogenous estrogens and progesterone in 10 premenopausal women using a gonadotropin-releasing hormone antagonist (GnRHa) for 10 days. On day 4 of GnRHa subjects received 0.1 mg of estradiol (GnRHa+E(2)), and on day 7 5 mg of MPA was added (GnRHa+E(2)+MPA). Endothelium-dependent vasodilation and endothelium-independent vasodilation of the brachial artery, lipids, homocysteine, high-sensitivity C-reactive protein, and endothelin-1 were assessed during treatment with GnRHa, GnRHa+E(2), and GnRHa+E(2)+MPA. Four additional subjects were tested to validate the efficacy of the GnRHa model and confirm the findings. Endothelium-dependent vasodilation was greater during GnRHa+E(2) than during GnRHa or GnRHa+E(2)+MPA (P = 0.006). Endothelin-1 was lower during GnRHa+E(2) than GnRHa alone (P = 0.039). Endothelin-1 increased with the addition of MPA and was not significantly different from GnRHa alone. There were no differences in the other markers of cardiovascular risk between hormone treatment days. These data suggest that acute MPA administration negates the beneficial effects of estradiol on endothelium-dependent vasodilation in young women. In addition, these data suggest that estradiol decreases endothelin-1 concentrations and the addition of MPA may counteract the effect of estradiol on endothelin-1.     

Deciduous Dentitions of Eocene Cebochoerid Artiodactyls and Cetartiodactyl Relationships

Deciduous lower premolars (milk teeth) of the Eocene artiodactyl family Cebochoeridae possess accessory denticles and are remarkably similar to both deciduous and adult teeth of the cetacean family Basilosauridae, suggesting that morphological characters of juvenile dentitions are important to understanding the phylogenetic origin of whales and morphological transitions in the cetartiodactyl lineage. Incorporation of these new characters into a larger phylogenetic analysis of morphological characters of artiodactyls, mesonychids, and basal and recent whales supports a monophyletic Cetartiodactyla, but does not directly support a whale–hippo relationship. However, the presence of accessory denticles on some artiodactyl dentitions weakens the morphological support for a monophyletic Artiodactyla, suggesting either that whales and cebochoerids may be more closely related than had been thought, or that cebochoerids share a developmental pathway with cetaceans.     

A gene-driven ENU-based approach to generating an allelic series in any gene

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F₁ ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.