Direct observation of glycogenesis and glucagon-stimulated glycogenolysis in the rat liver in vivo by high-field carbon-13 surface coil NMR

High-field 13C surface coil nuclear magnetic resonance has been employed to investigate glucose and glycogen metabolism in rat liver in vivo. Natural abundance and isotopically enriched proton-decoupled 13C NMR experiments were conducted at 90.56 MHz on a standard commercial spectrometer utilizing a laboratory-built high-sensitivity double-resonance coaxial coil probe. At variance with a previous preliminary report, natural abundance spectra of the liver in vivo from a rat fed ad libitum reveal resonances of substantial intensity from hepatic glycogen with approximately 10 min of signal averaging. The response of hepatic glycogen levels to an intravenous injection of the hormone glucagon was continuously monitored through the glycogen C-1 carbon resonance intensity; this revealed an average 60% depletion of hepatic glycogen stores in vivo within approximately 1 h. In a complementary study utilizing fasted rats, 100 mg of D-[1-13C]glucose (90% enriched) was administered via a peripheral vein injection and continuously monitored by 13C NMR with 3-min time resolution as it was incorporated into hepatic glycogen. The C-1 carbon resonances of hepatic glucose and glycogen are well-resolved in vivo enabling the time course for the relative change in concentration for both metabolites to be established simultaneously. The 13C label incorporated into the glycogen pool reaches a steady-state level in approximately 40 min.     

Factors affecting in vitro propagation of Yucca glauca

A micropropagation system was developed to facilitate the release and subsequent testing of unique pink- or white-flowered selections of Yucca glauca. Shoot tip explants from mature plants were cultured on Murashige and Skoog medium supplemented with factorial combinations of -naphthaleneacetic acid (NAA) (0.0 to 3.2 M) and 6-benzylaminopurine (BA) (0.0 to 45 M). Shoots were found to proliferate with increasing concentrations of BA and to produce callus and poorer quality shoots in the presence of NAA and the absence of BA. The response to BA and NAA was similar among 3 genotypes examined. A comparison of BA and N₆-(₂-isopentenyladenine) (2iP) showed that 2iP was not effective in promoting shoot proliferation. Shoot tips rooted in the absence of growth regulators or in the presence of low concentrations of indole-3-butyric acid (IBA). Plantlets were successfully acclimated to greenhouse conditions.     

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.     

Simultaneous In Vivb Monitoring of Cerebral Deoxyglucose and Deoxyglucos-6-Phosphate by l3C{lH} Nuclear Magnetic Resonance Spectroscopy

The capacity of brain to dephosphorylate glucose-6-phosphate has been established, but the magnitude and significance of this capacity in vivo are debated, particularly in regard to dephosphorylation of the glucose analog 2-deoxyglucose. We now report results of external measurement in the brains of conscious rats with simultaneous resolution and quantification of both 2-deoxyglucose and its phosphorylated product by nuclear magnetic resonance (NMR) techniques that used 2-[6-13]deoxyglucose together with proton-decoupled 13C surface-coil spectroscopy. As NMR techniques require large doses of 2-deoxyglucose, a dose comparison was first made using decay curves of total label after tracer doses of 2-[14C]deoxyglucose without versus with unlabeled deoxyglucose at 500 mg/kg (the NMR dose). Similar cerebral half-lives for the two doses were found, and no behavioral evidence for toxicity of the NMR dose was seen. In vivo NMR monitoring of conscious rats showed that the analog reached maximal cerebral concentration within 10 min of the intravenous bolus and decayed with a half-life of 29 +/- 7 min (n = 4; mean +/- SEM), whereas 2-deoxyglucose-6-phosphate reached peak concentration between 30 and 40 min and decayed with a half-life of 2.1 +/- 0.3 h, equivalent to a fractional loss of 0.8%/min. Thirty-one percent (+/- 5%) of the total analog pool (which showed a half-life of 1.4 h) consisted of 2-deoxyglucose at 45 min after the bolus. The results support an active but limited role for dephosphorylation by normal brain in glucose analog (and potentially glucose) metabolism in the unstimulated conscious rat and a wide concentration range for the metabolic operations involved.     

Highly avid magnetic bead capture: An efficient selection method for de novo protein engineering utilizing yeast surface display

Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million‐fold improvements in binding affinity by alternating rounds of diversification and flow cytometry‐based selection. However, flow cytometry is not well suited for isolating de novo binding clones from naïve libraries due to limitations in the size of the population that can be analyzed, the minimum binding affinity of clones that can be reliably captured, the amount of target antigen required, and the likelihood of capturing artifactual binders to the reagents. Here, we demonstrate a method for capturing rare clones that maintains the advantages of yeast as the expression host, while avoiding the disadvantages of FACS in isolating de novo binders from naïve libraries. The multivalency of yeast surface display is intentionally coupled with multivalent target presentation on magnetic beads—allowing isolation of extremely weak binders from billions of non‐binding clones, and requiring far less target antigen for each selection, while minimizing the likelihood of isolating undesirable alternative solutions to the selective pressure. Multivalent surface selection allows 30,000‐fold enrichment and almost quantitative capture of micromolar binders in a single pass using less than one microgram of target antigen. We further validate the robust nature of this selection method by isolation of de novo binders against lysozyme as well as its utility in negative selections by isolating binders to streptavidin‐biotin that do not cross‐react to streptavidin alone. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Arctic shrub growth trajectories differ across soil moisture levels

The circumpolar expansion of woody deciduous shrubs in arctic tundra alters key ecosystem properties including carbon balance and hydrology. However, landscape‐scale patterns and drivers of shrub expansion remain poorly understood, inhibiting accurate incorporation of shrub effects into climate models. Here, we use dendroecology to elucidate the role of soil moisture in modifying the relationship between climate and growth for a dominant deciduous shrub, Salix pulchra, on the North Slope of Alaska, USA. We improve upon previous modeling approaches by using ecological theory to guide model selection for the relationship between climate and shrub growth. Finally, we present novel dendroecology‐based estimates of shrub biomass change under a future climate regime, made possible by recently developed shrub allometry models. We find that S. pulchra growth has responded positively to mean June temperature over the past 2.5 decades at both a dry upland tundra site and an adjacent mesic riparian site. For the upland site, including a negative second‐order term in the climate–growth model significantly improved explanatory power, matching theoretical predictions of diminishing growth returns to increasing temperature. A first‐order linear model fit best at the riparian site, indicating consistent growth increases in response to sustained warming, possibly due to lack of temperature‐induced moisture limitation in mesic habitats. These contrasting results indicate that S. pulchra in mesic habitats may respond positively to a wider range of temperature increase than S. pulchra in dry habitats. Lastly, we estimate that a 2°C increase in current mean June temperature will yield a 19% increase in aboveground S. pulchra biomass at the upland site and a 36% increase at the riparian site. Our method of biomass estimation provides an important link toward incorporating dendroecology data into coupled vegetation and climate models.     

Chronobiologic Factors in Experimental Stress Ulcer

The available literature on chronobiologic factors in experimental stress ulcer is extremely small and thematically limited. It focuses almost exclusively on circadian rhythms and, within that, on rhythms related to light-dark cycles, activity and body temperature. Among these, only differences in ulcer induction related to circadian activity patterns have been adequately demonstrated. Other circadian patterns and other temporal phase relationships might be profitably explored, including those related to postnatal development. It is also likely that the important relationships between biorhythms and stress ulcer are not limited to ulcer induction. Future studies should address chronobiologic factors in predisposition, severity of illness, the probability of recovery and response to various therapeutic interventions.     

THE EFFECTIVENESS OF FUNGUS GNATS AS POLLINATORS

Fungus gnats (Sciaridae and Mycetophilidae) are the principal pollinators of Listera cordata (L.) R. Br. (Orchidaceae) and Scoliopus bigelovii Torr. (Liliaceae) in coastal redwood forests of northern California. Although primitive diptera have generally been regarded as relatively inefficient pollinators, fruit set for both species is high: 61–78% for L. cordata (1976–1978) and 94.3–98.5% for S. bigelovii (1978–1979). Since probability of pollination per visit is low, we attribute high fruit set to the large number of gnats present at our study sites and corresponding large number of visits to flowers. The relative frequency of geitonogamous vs. xenogamous pollen flow was estimated by emasculating flowers and subsequently comparing pollen reception or fruit set of emasculates with controls. Results for both species indicate that interplant movement of pollen is common. Thus, fungus gnats can be effective pollen vectors, both in terms of overall fruit set and potential for cross-pollination.