Speciation in chestnut-shouldered fairy-wrens (Malurus spp.) and rapid phenotypic divergence in variegated fairy-wrens (Malurus lamberti): a multilocus approach.

The chestnut-shouldered fairy-wrens comprise a subgroup of four species in the genus Malurus (Passeriformes: Maluridae). Collectively, they are widespread across the Australian continent but phenotypic variation is strongly structured geographically in just one species, M. lamberti. Earlier phylogenetic analyses of this group have been limited to one or two individuals for each species and have not represented all currently recognised subspecies of M. lamberti. Historically, the taxonomy and nomenclature of the M. lamberti complex has been debated, in part because of morphological similarities among its subspecies and another member of the group, M. amabilis. We reconstructed the phylogeny of all four species of chestnut-shouldered fairy-wrens including all four subspecies of M. lamberti using a mitochondrial gene (ND2), five anonymous nuclear loci and three nuclear introns. Phylogenetic analysis of the mitochondrial ND2 gene nests M. amabilis within M. lamberti rendering the latter paraphyletic. Individual nuclear gene trees failed to reliably resolve each of the species boundaries or the phylogenetic relationships found in the mtDNA tree. When combined, however, a strongly supported overall topology was resolved supporting the monophyly of M. lamberti and its sister species relationship to M. amabilis. Current subspecific taxonomy of M. lamberti was not concordant with all evolutionary lineages of M. lamberti, nominotypical M. l. lamberti being the only subspecies recovered as a monophyletic group from mtDNA. Some genetic structuring is evident and potential barriers to gene flow are discussed.

Self-regulation of driving and its relationship to driving ability among older adults.

Although it is known that older drivers limit their driving, it is not known whether this self-regulation is related to actual driving ability. A sample of 104 older drivers, aged between 60 and 92, completed a questionnaire about driving habits and attitudes. Ninety of these drivers also completed a structured on-road driving test. A measure of self-regulation was derived from drivers' self-reported avoidance of difficult driving situations. The on-road driving test involved a standard assessment used to determine fitness to drive. Driving test scores for the study were based on the number of errors committed in the driving tests, with weightings given according to the seriousness of the errors. The most commonly avoided difficult driving situations, according to responses on the questionnaire, were parallel parking and driving at night in the rain, while the least avoided situation was driving alone. Poorer performance on the driving test was not related to overall avoidance of difficult driving situations. Stronger relationships were found between driving ability and avoidance of specific difficult driving situations. These specific driving situations were the ones in which the drivers had low confidence and that the drivers were most able to avoid if they wished to.

31P MAS-NMR of human erythrocytes: independence of cell volume from angular velocity.

31P magic angle spinning NMR (MAS-NMR) spectra were obtained from suspensions of human red blood cells (RBCs) that contained the cell-volume-sensitive probe molecule, dimethyl methylphosphonate (DMMP). A mathematical representation of the spectral-peak shape, including the separation and width-at-half-height in the 31P NMR spectra, as a function of rotor speed, enabled us to explore the extent to which a change in cell volume would be reflected in the spectra if it occurred. We concluded that a fractional volume change in excess of 3% would have been detected by our experiments. Thus, the experiments indicated that the mean cell volume did not change by this amount even at the highest spinning rate of 7 kHz. The mean cell volume and intracellular 31P line-width were independent of the packing density of the cells and of the initial cell volume. The relationship of these conclusions to other non-NMR studies of pressure effects on cells is noted.

G protein-coupled receptor fusion proteins in drug discovery.

A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein alpha subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist/inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.

The effect of aging on the immunohistochemistry of apolipoprotein E in the liver.

Apolipoprotein E (apoE) is involved in hepatic disposition of chylomicron remnants, which is impaired in old age. Isoforms of apoE have been implicated in age-related diseases and possibly the aging process itself. Because the effects of old age on expression and distribution of apoE in the liver have not been reported, we studied the effect of old age on the immunohistochemistry of apoE in the livers of humans and the non-human primate, Papio hamadryas. Overall, old age was not associated with marked changes in the expression of apoE between adult (48+/-19 years) and old (82+/-5 years) humans. However, there was a change in the distribution of apoE staining. The livers of older humans displayed increased hepatocyte cytoplasmic staining and reduced peri-sinusoidal staining. Similar trends were noted in the livers from the baboons. Such findings are suggestive of altered apoE recycling in old age and have implications for age-related dyslipidaemia.

Wash-in methodology and modeling to determine hepatocellular D-glucose transport in the perfused rat liver.

Wash-in experiments, which may be useful for the study of the disposition of substrates in the liver, have not been well described. To investigate physiological models for wash-in curves, we performed experiments on the perfused livers of male Wistar rats anesthetized with pentobarbital. Test perfusate contained (14)C-sucrose as the extracellular marker and (3)H-glucose. Liver perfusions were performed with background glucose concentrations of 5.7, 10.7, 51.2, or 108.5 mM. Outflow time-activity curves were analyzed with the use of four models. The V(max) and K(m) for the influx of glucose were 1.1 +/- 0.03 micromol/s/g and 41 +/- 3 mM with the Crone-Renkin early extraction model; 1.4 +/- 0.04 micromol/s/g and 36 +/- 3 mM with dispersion model analysis; 1.8 +/- 0.1 micromol/s/g and 25 +/- 4 mM with the Goresky distributed model to fit differentiated wash-in curves; and 2.7 +/- 0.6 micromol/s/g and 28 +/- 21 mM with compartmental analysis. There was reasonable agreement between the four models, and they yielded results similar to those reported for glucose uptake in other preparations.

Carbon monoxide disposition and permeability-surface area product in the foetal circulation of the perfused term human placenta.

In order to estimate the placental barrier to gas transfer, a novel carbon monoxide (CO) wash-in method was used to estimate the permeability-surface area (PS) product for the transfer of gas across the foetal circulation in the perfused human term placenta. The PS product for CO was 0.0096+/-0.006 ml/s/g or 0.012+/-0.007 ml/s/g using compartmental or Crone-Renkin analysis, respectively. Using this result and a published estimate of the placental capillary surface area, the permeability coefficient to CO across the foetal circulation was found to be approximately 4 x 10(-5)cm/s. This result is compatible with the hypothesis that the foetal circulation of the human placenta imposes a potentially significant barrier to gas transfer.

Cell membrane transport of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the liver and systemic bioavailability.

Modulation of hepatic disposition of MPTP could influence susceptibility to its neurotoxicity. Therefore, we studied hepatocellular transport of MPTP in the perfused rat liver and isolated rat hepatocytes. The perfused liver extensively extracted MPTP. Amiloride and tubocurarine, inhibitors of OCT1, increased MPTP recovery (253 +/- 78 and 283 +/- 64%, respectively) and reduced PS(influx) (0.69 +/- 0.36 to 0.27 +/- 0.11, and 0.97 +/- 0.50 to 0.23 +/- 0.05 ml/s/g, respectively). P-glycoprotein inhibitor, daunomycin, and Oatp 1 & 2 inhibitor, rifamycin, had no effect. In isolated hepatocytes, amiloride and tubocurarine increased hepatic uptake of MPTP (23 +/- 12 and 6 +/- 2%, respectively). Daunomycin reduced MPTP uptake by 22 +/- 8% and rifamycin had no effect. Only a small proportion of MPTP is taken up into hepatocytes by transporters; however, modulation of these transport mechanisms will influence systemic bioavailability.

Pseudocapillarization and associated energy limitation in the aged rat liver.

Age-related impairment of drug metabolism by the liver is consistent with hepatocyte hypoxia, suggestive of the development of a diffusional barrier to oxygen supply. Because the effects of aging on the diffusional pathway (sinusoidal endothelium and space of Disse) have not been described, we performed comparative studies on the livers of Fischer F344 rats aged 4 to 7, 12 to 15, and 24 to 27 months. Light-microscopic examination revealed no evidence of fibrosis, cirrhosis, or other specific pathology. In contrast, scanning and transmission electron-microscopic examination revealed that aging is associated with pseudocapillarization of the sinusoidal endothelium, indicated by defenestration with reduced porosity, thickening of the endothelium, infrequent development of basal lamina, and only minor collagen deposits in the space of Disse. Furthermore, immunohistochemistry studies showed strong expression of collagen IV, moderate expression of factor VIII-related antigen, and weak expression of collagen I along the sinusoids of livers from old rats (P <.0001). In vitro (31)P magnetic resonance spectroscopy analysis showed that aging is associated with changes in high-energy phosphate and other metabolites, consistent with hepatocyte hypoxia. Aging in the liver is associated with changes in the sinusoidal endothelium and space of Disse that may restrict the availability of oxygen and other substrates.

Carbon monoxide wash-in method to determine gas transfer in vascular beds: application to rat hindlimb.

The vascular barrier to gas transfer is an important physiological parameter; however, no readily applicable technique exists to quantitate the process. A simple technique to measure the permeability-surface area (PS) product for gas transfer in vascular beds is proposed using wash in of carbon monoxide (CO) and Crone-Renkin analysis. Wash-in experiments were performed on the perfused hindlimbs of male Wistar rats (n = 15) by using CO as a surrogate marker for oxygen and technetium-99m-labeled albumin as the vascular marker. The use of CO and erythrocyte-free perfusate and the collection of outflow samples into tubes preloaded with erythrocytes obviated the need for an anaerobic collection device or consideration of Hb binding in the analysis. The PS product for CO was determined from the early extraction as 0.013 +/- 0.006 ml. s(-1). g(-1). Compartmental analysis revealed that the fractional recovery of CO was 0.45 +/- 0.14 and the volume of distribution was 2.31 +/- 0.76 ml/g. This technique detected a small measurable barrier to the transfer of CO across the hindlimb vasculature and is potentially applicable to other vascular beds in health and disease.

The effect of acute oxidative stress on the ultrastructure of the perfused rat liver.

Ageing and liver disease are associated with ultrastructural changes in the hepatic sinusoid. Because of the possibility that reactive oxygen species could mediate these processes, we examined the effect of acute oxidative stress on the ultrastructure of the intact liver. Rat livers were perfused ex vivo, in situ with hydrogen peroxide via the portal vein. The livers were then fixed and the ultrastructure of the liver tissue examined with transmission and scanning electron microscopy. The effects of hydrogen peroxide were largely confined to the perisinusoidal areas. The sinusoidal endothelial cells became swollen and more porous, with large gaps replacing sieve plates. The space of Disse showed an increase in volume and the density of hepatocyte projections decreased. Kupffer cell activation was noted. Little or no ultrastructural change was observed within the hepatocytes. Oxidative stress delivered via the portal vein dramatically alters the ultrastructure of the perisinusoidal regions of the liver. This process may contribute to the pathogenesis of disease and age-related changes in the liver.

Hydrogen abstraction from ionic liquids by benzophenone triplet excited states.

The activation energy for hydrogen abstraction from imidazolium-based ionic liquids is significantly higher than that observed in conventional solvents.

Ligand regulation of green fluorescent protein-tagged forms of the human beta(1)- and beta(2)-adrenoceptors; comparisons with the unmodified receptors.

Stable clones of HEK293 cells expressing either FLAG(TM) epitope-tagged, wild type human beta(1)- and beta(2)-adrenoceptors or C-terminally green fluorescent protein (GFP)-tagged forms of these receptors were established. The binding affinity of [(3)H]-dihydroalprenolol and other ligands was little affected by addition of GFP to the C-terminal of either receptor. Isoprenaline induced the internalisation of both beta(1)-adrenoceptor-GFP and beta(2)-adrenoceptor-GFP and following removal of the agonist both constructs were able to recycle to the cell surface. The extent of internalisation of beta(2)-adrenoceptor-GFP produced by isoprenaline was substantially greater than for beta(1)-adrenoceptor-GFP. C-terminal addition of GFP slowed markedly the rate of internalization of both the beta(1)-adrenoceptor and the beta(2)-adrenoceptor in response to isoprenaline. Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native beta(2)-adrenoceptor compared to beta(2)-adrenoceptor-GFP although both were equally effectively removed from the plasma membrane. Sustained exposure to isoprenaline resulted in a large fraction of beta(2)-adrenoceptor-GFP becoming trapped in internal vesicles/lysosomes but not degraded. Even after sustained exposure to isoprenaline a significant fraction of beta(1)-adrenoceptor-GFP remained at the cell surface. These results indicate that although GFP tagging of beta-adrenoceptors can provide qualitative visual patterns of agonist-induced receptor trafficking and regulation in HEK293 cells the quantitative details vary markedly from those obtained with the unmodified receptors.

Acute oxygen supplementation restores markers of hepatocyte energy status and hypoxia in cirrhotic rats.

The oxygen limitation hypothesis states that hepatocyte hypoxia is the mechanism determining metabolic restriction in the cirrhotic liver. Therefore we studied markers of hepatocyte energy state and cellular hypoxia in livers of normal and cirrhotic rats before and after oxygen supplementation. Rats with carbon tetrachloride-induced cirrhosis and procedural control rats were exposed to either room air or a hyperoxic gas mixture for 1 h immediately before freeze clamping and perchloric acid extraction of liver tissue. Extracts were assessed by (31)P NMR and enzymatic assays. Livers from cirrhotic rats breathing room air showed a reduced ratio of ATP/ADP, an increased ratio of inorganic phosphate/ATP, and a trend toward an increased ratio of lactate/pyruvate compared with procedural control livers (ATP/ADP 1.73 +/- 0.35 versus 2.68 +/- 0.61, P <.05; P(i)/ATP 2.74 +/- 0.48 versus 1.56 +/- 0.26, P <.05; lactate/pyruvate 29.3 +/- 6.4 versus 22.5 +/- 7.4, P =.18). After supplementation with oxygen for 1 h, these ratios in cirrhotic livers approached control values. A variety of other metabolic markers affected by cirrhosis showed variable trends toward normal in response to oxygen supplementation, whereas minor trends toward an increase in ATP levels in control animals suggest the possibility of marginal oxygen limitation in normal livers. The data are consistent with the hypothesis that hepatocytes in cirrhotic livers have normal metabolic capacity but are constrained by a deficit in oxygen supply. Interventions aimed at increasing oxygen supply to the liver may have both short- and long-term therapeutic value in the management of cirrhosis.

Hepatic disposition of neurotoxins and pesticides.

The hepatic disposition of pesticides and neurotoxins may influence susceptibility to Parkinson's disease. Therefore we examined the behaviour of paraquat, dichlorodiphenyltrichloroethane (DDT), malathion and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in perfused rat liver using the multiple indicator-dilution technique. The values for the recovery of paraquat, DDT, malathion and MPTP were 1.05+/-0.12, 0.32+/-0.01, 0.11+/-0.02 and 0.02+/-0.01, respectively. The volumes of distribution were 0.28+/-0.13, 0.69+/-0.12, 3.30+/-0.58 and 5.10+/-6.00 ml/g, respectively. The permeability-surface area products suggest that transport of DDT and MPTP across cell membranes is by simple diffusion. However, there may be a specific influx mechanism for malathion and a specific efflux mechanism for paraquat. There is considerable variability in the hepatic disposition of putative neurotoxins such as MPTP and pesticides. Factors that influence the hepatic disposition of neurotoxins may alter susceptibility to neurotoxic diseases however the effects will be diverse.

Oxidative injury reproduces age-related impairment of oxygen-dependent drug metabolism.

The Oxygen Diffusion Barrier Hypothesis states that aging in the liver is associated with restricted oxygen uptake that explains the age-related impairment of phase I drug clearance observed in vivo with preservation of in vitro phase I enzyme activity and in vivo phase II drug clearance. Aging in the liver may be secondary to oxidative stress. Therefore we examined the effects of oxidative injury on oxygen uptake, and phase I and phase II drug metabolism in the liver. Oxidative stress was induced in the perfused rat liver with hydrogen peroxide. The intrinsic clearances of propranolol and morphine were used as markers of phase I and phase II activity, respectively. Oxidative injury was associated with a 14+/-99% (P=0.03) reduction in oxygen uptake. The decrease in the intrinsic clearance of propranolol was greater than that of morphine (57+/-14% vs 34+/-7% P<0.005). This result supports the concept of a restriction of oxygen supply constraining hepatic drug metabolism following oxidative stress. This has implications for aging and hepatic drug metabolism.

Cephalosporin clinical concentration-time profile modelling and in-vitro bactericidal effects on Escherichia coli.

We assessed the cephalosporin concentration-time curve area (AUC), peak concentration, maintained concentration and duration of exposure on in-vitro bactericidal effects on Escherichia coli NCTC 10418, using exposures modelling cephazolin clinical profiles after 1 g and 2 g i.m. injection, equal AUC exposures (288 mg x h/L, 576 mg x h/L; 48 h) and constant exposures to 6, 12 and 24 mg/L. Cephalosporin dosage exposures based on maintenance of concentrations at multiples (6-24 times) of the MIC were not as effective in early or sustained (24 h) bactericidal effect as exposures modelling im injection profiles with equal or lower AUC (P<0.05, ANOVA). Similar results applied to i.m. comparisons with equal AUC exposures modelling extremes of concentration and time exposures. These results indicate a need for intermittent dosage to produce optimally effective profiles, and raise the possibility that these optimum dosing profiles may allow an extension of minimum interdose intervals beyond 8 h.