High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin

An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37°C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) is formed. The metabolite was analyzed by reversed-phase HPLC with fluorescence detection. The limits of detection of the metabolite were 5.0 fmol per injection, a sensitivity at least one order of magnitude greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited.     

Influence of drainage divides versus arid corridors on genetic structure and demography of a widespread freshwater turtle,Emydura macquarii krefftii,from Australia

The influence of Pleistocene climatic cycles on Southern Hemisphere biotas is not yet well understood. Australia's eastern coastal margin provides an ideal setting for examining the relative influence of landscape development, sea level fluctuation, and cyclic climatic aridity on the evolution of freshwater biodiversity. We examined the impact of climatic oscillations and physical biogeographic barriers on the evolutionary history of the wide‐ranging Krefft's river turtle (Emydura macquarii krefftii), using range‐wide sampling (649 individuals representing 18 locations across 11 drainages) and analysis of mitochondrial sequences (~1.3‐kb control region and ND4) and nuclear microsatellites (12 polymorphic loci). A range of phylogeographic (haplotype networks, molecular dating), demographic (neutrality tests, mismatch distributions), and population genetic analyses (pairwise F_ST, analysis of molecular variance, Bayesian clustering analysis) were implemented to differentiate between competing demographic (local persistence vs. range expansion) and biogeographic (arid corridor vs. drainage divide) scenarios. Genetic data reveal population genetic structure in Krefft's river turtles primarily reflects isolation across drainage divides. Striking north‐south regional divergence (2.2% ND4 p‐distance; c. 4.73 Ma, 95% higher posterior density (HPD) 2.08–8.16 Ma) was consistent with long‐term isolation across a major drainage divide, not an adjacent arid corridor. Ancient divergence among regional lineages implies persistence of northern Krefft's populations despite the recurrent phases of severe local aridity, but with very low contemporary genetic diversity. Stable demography and high levels of genetic diversity are inferred for southern populations, where aridity was less extreme. Range‐wide genetic structure in Krefft's river turtles reflects contemporary and historical drainage architecture, although regional differences in the extent of Plio–Pleistocene climatic aridity may be reflected in current levels of genetic diversity.     

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.     

Lipid rafts regulate lipopolysaccharide-induced activation of Cdc42 and inflammatory functions of the human neutrophil

Lipid rafts are cholesterol-rich membrane microdomains that are thought to act as coordinated signaling platforms by regulating dynamic, agonist-induced translocation of signaling proteins. They have been described to play a role in multiple prototypical cascades, among them the lipopolysaccharide pathway, and to host multiple signaling proteins, including kinases and low molecular weight G-proteins. Here we report lipopolysaccharide-induced activation of the Rho family GTPase Cdc42, and we show its activation in the human neutrophil to be mediated by a p38 mitogen-activated protein kinase-dependent mechanism. Subcellular fractionation reveals that lipopolysaccharide induces translocation of Cdc42 to lipid rafts, where it and p38 are both found to be activated. By contrast, lipopolysaccharide causes translocation of Rac from the polymorphonuclear leukocyte (PMN) rafts and does not induce its activation. With the use of methyl-beta-cyclodextrin, a cholesterol-depleting agent that reversibly disrupts rafts, we confirm an important regulatory role for rafts in the activation state of p38 and Cdc42 and in the Rho GTPase-dependent functions superoxide anion production and actin polymerization. Methyl-beta-cyclodextrin induces activation of p38 and Cdc42, but not Rac, in the nonstimulated PMN, yet inhibits subsequent lipopolysaccharide-induced activation of p38 and Cdc42. In parallel, methyl-beta-cyclodextrin primes the human PMN for subsequent superoxide release triggered by the formylated bacterial tripeptide formyl-Met-Leu-Phe, and induces actin polymerization in a subcellular distribution distinct from that induced by lipopolysaccharide. In sum, these findings provide evidence for an important regulatory role of cholesterol in both transmission of the lipopolysaccharide signal and the inflammatory phenotype of the human neutrophil.     

Life History Variation in Female Gambusia hubbsi

Gambusia hubbsi populations occur in a variety of fresh and brackish-water habitats on Andros, Bahamas. These include shallow water sites (tidal creeks, lakes, roadside ditches), and blueholes (vertical solution caves). In some blueholes G. hubbsi is the only species present, in others it co-occurs with other species, principal among these is a predator, Eleotris pisonis. By contrast to blueholes, shallow water sites have highly variable temperature and depth. In addition, they are frequented by avian piscivores and may be occasionally occupied by piscivores such as Eleotris. We sampled 10 shallow water sites, 14 blueholes where Eleotris is absent and 12 blueholes where Eleotris co-occurs with G. hubbsi. We measured and compared variation in female body size, fecundity, and reproductive investment among these three habitats. The observed patterns of life history variation are only partially in accord with expectations from theory regarding the effects of predation and seasonality on life history variation. Samples from populations that colonized a series of man-made trenches (Well Fields), a set of introductions into that habitat, and changes in life history traits of lab-raised females from three blueholes, suggest that the observed pattern of life history variation in other habitats also reflects differences in food availability among habitats, and imperfectly reflects the potential phenotypic variability of this species.     

Human megakaryocytic progenitors (CFU-M) assayed in methylcellulose: Physical characteristics and requirements for growth

The basic culture requirements and several physical characteristics were defined for megakaryocytic colony-forming cells (CFU-M) from normal human marrow growing in methylcellulose. Ficoll-hypaque separated mononuclear cells from human, marrow gave rise to megakaryocytic colonies in the presence of normal human plasma and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Their identity as megakaryocytic colonies was confirmed by immunofluorescence staining with a monoclonal antibody to human factor VIII antigen and by electron microscopy of individually harvested colonies. Demonstration of the single-cell origin of the colonies was provided by analysis of the glucose-6-phosphate dehydrogenase (G-6-PD) enzyme type of individually harvested colonies grown from a G-6-PD heterozygote. The colonies grew best in heparinized or citrated plasma as opposed to serum. Detailed studies suggested that platelet-release products were responsible for this difference. Tritiated thymidine suicide studies showed that the percentage of CFU-M in DNA synthesis was 23 ± 8% (n = 10). The modal velocity sedimentation rate of CFU-M was 4.9 ± 0.6 mm/hr (n = 4) while that of concurrently studied granulocyte/macrophage colony-forming cells (CFU-GM) was 5.7 ± 0.5 mm/hr. Examination of the PHA-LCM dose-response characteristics suggested the presence in the conditioned medium of an inhibitor to megakaryocyte colony growth which was partially removed by chromatography of the medium on Sephadex G-100. The resulting conditioned medium increased the cloning efficiency for CFU-M compared with that with crude PHA-LCM (15.3 ± 7.0 and 8.2 ± 5.3/10⁵ marrow cells, respectively).     

Activation of the 12-lipoxygenase and signal transducer and activator of transcription pathway during neointima formation in a model of the metabolic syndrome

Insulin resistance (IR) is associated with an increased risk of cardiovascular diseases. The obese Zucker rat (ZR) is a model of IR that shows markedly increased insulin and triglyceride concentrations without major changes in glucose. In this study, we evaluated the response of obese and lean ZR to carotid balloon injury and determined potential mechanisms and treatments. The neointima-to-media ratio of obese ZR was greater than that of lean ZR, starting at 14 days after injury, and persisted until at least day 30. An enhanced inflammatory response to balloon injury in the obese ZR was reflected by significantly higher ED1-positive macrophage cells in the injured vessel wall compared with that in lean ZR at 3, 7, and 14 days after balloon injury. Inflammatory mediators 12-lipoxygenase (12-LO) and STAT4 were studied in neointimal lesions. Expression of 12-LO RNA was increased beginning at day 7 and showed increases of 4.3-fold on day 14 and 7-fold on day 30 in obese ZR compared with lean animals. Staining of phosphorylated STAT4 (PSTAT4), the activated form of STAT4, in lesions from obese ZR was also increased compared with that in leans. We tested the effects of a novel anti-inflammatory agent, lisofylline (LSF), in the obese ZR. LSF markedly reduced neointimal formation in the obese ZR. LSF also reduced monocyte/macrophage infiltration into the vessel wall and the activation of PSTAT4. These studies suggest both the presence of an exaggerated injury response in the insulin-resistant obese ZR model and that inflammation plays a major role in mediating neointimal growth.     

By binding SIRPalpha or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation

Surfactant proteins A and D (SP-A and SP-D) are lung collectins composed of two regions, a globular head domain that binds PAMPs and a collagenous tail domain that initiates phagocytosis. We provide evidence that SP-A and SP-D act in a dual manner, to enhance or suppress inflammatory mediator production depending on binding orientation. SP-A and SP-D bind SIRPalpha through their globular heads to initiate a signaling pathway that blocks proinflammatory mediator production. In contrast, their collagenous tails stimulate proinflammatory mediator production through binding to calreticulin/CD91. Together a model is implied in which SP-A and SP-D help maintain a non/anti-inflammatory lung environment by stimulating SIRPalpha on resident cells through their globular heads. However, interaction of these heads with PAMPs on foreign organisms or damaged cells and presentation of the collagenous tails in an aggregated state to calreticulin/CD91, stimulates phagocytosis and proinflammatory responses.