The importance of membrane integrity in kinetic characterizations of the microsomal glucose-6-phosphatase system

The transport model of glucose-6-phosphatase (EC 3.1.3.9) was recently challenged by a report that detergent treatment had no effect on the presteady state kinetics of glucose-6-P hydrolysis catalyzed at 0 degree C by the enzyme in liver microsomes previously frozen in 0.25 M mannitol (Zakim, D., and Edmondson, D. E. (1982) J. Biol. Chem. 257, 1145-1148). The lack of response to detergent is shown to be the expected consequence of the conditions used in the presteady state measurements. First, when the assay temperature was reduced from 30 to 0 degree C the depression in the glucose-6-P phosphohydrolase activity of intact microsomes (i.e. the system) was much greater than that of fully disrupted microsomes (i.e. enzyme). This indicates that temperature influences transport much more than hydrolysis of glucose-6-P. As a result, the contribution of a small fraction of enzyme associated with disrupted structures is markedly exaggerated, so it becomes the predominant hydrolytic activity before detergent treatment. Second, freezing microsomes in 0.25 M mannitol caused such extensive disruption that all of the activity manifest at 0 degree C could be attributed to enzyme in disrupted structures. The present findings underscore the importance of assessing the state of intactness of "untreated" microsomes and quantifying the contribution of the disrupted component in kinetic analyses of the glucose-6-phosphatase system. The proposition that the detergent-induced changes in the kinetic properties of glucose 6-phosphatase represent removal of constraints imposed on the enzyme by the membrane environment rather than increased access of enzyme to substrate is critically analyzed.     

Inconsistent detection of extinction debts using different methods

The extinction debt, delayed species extinctions following landscape degradation, is a widely discussed concept. But a consensus about the prevalence of extinctions debts is hindered by a multiplicity of methods and a lack of comparisons among habitats. We applied three contrasting species–area relationship methods to test for plant community extinction debts in three habitats which had different degradation histories over the last century: calcareous grassland, heathland and woodland. These methods differ in their data requirements, with the first two using information on past and current habitat area alongside current species richness, whilst the last method also requires data on past species richness. The most data‐intensive, and hence arguably most reliable method, identified extinction debts across all habitats for specialist species, whilst the other methods did not. All methods detected an extinction debt in calcareous grassland, which had undergone the most severe degradation. We conclude that some methods failed to detect an extinction debt, particularly in habitats that have undergone moderate degradation. Data on past species numbers are required for the most reliable method; as such data are rare, extinction debts may be under‐reported.     

From Mesolithic hunters to Iron Age herders: a unique record of woodland use from eastern central Europe (Czech Republic)

Vegetation History and Archaeobotany - In a continuous, perfectly stratified sedimentary sequence which was discovered under a large sandstone overhang in northern Bohemia, Czech Republic, we...     

In vitro Inhibition of Clinical Isolates of Otitis Media Pathogens by the Probiotic Streptococcus salivarius BLIS K12

Otitis media is a common childhood infection, frequently requiring antibiotics. With high rates of antibiotic prescribing and increasing antibiotic resistance, new strategies in otitis media prevention and treatment are needed. The aim of this study was to assess the in vitro inhibitory activity Streptococcus salivarius BLIS K12 against otitis media pathogens. Efficacy of the bacteriocin activity of S. salivarius BLIS K12 against the otitis media isolates was assessed using the deferred antagonism test. Overall, 48% of pathogenic isolates exhibited some growth inhibition by S. salivarius BLIS K12. S. salivarius BLIS K12 can inhibit the in vitro growth of the most common pathogens.

     

Within-laboratory and between-laboratory variability in the measurement of anti-müllerian hormone determined within an external quality assurance scheme

Ten laboratories in an external quality assurance scheme used the same assay to measure anti-müllerian hormone concentration (Beckman Coulter Gen II) and received twenty serum samples distributed over a 15 month period. The mean bias for all results was only ?0.089%, but there was large coefficient of repeatability of 38.8% (sample bias ranged from ?37.9% to +54.7%). While each laboratory showed good reproducibility, there was a wide range of average values relative to the consensus value from ?24.0% to +22.7%. This between-laboratory variability suggests clinicians should use the same laboratory to avoid problems with result interpretation.     

Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite,Caloric Intake and Fat Storage in Drosophila

Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C_14:0) and ceramide diene (C_14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C_14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.     

Climate change,extinction, and Sky Island biogeography in a montane lizard

Around the world, many species are confined to “Sky Islands,” with different populations in isolated patches of montane habitat. How does this pattern arise? One scenario is that montane species were widespread in lowlands when climates were cooler, and were isolated by local extinction caused by warming conditions. This scenario implies that many montane species may be highly susceptible to anthropogenic warming. Here, we test this scenario in a montane lizard (Sceloporus jarrovii) from the Madrean Sky Islands of southeastern Arizona. We combined data from field surveys, climate, population genomics, and physiology. Overall, our results support the hypothesis that this species' current distribution is explained by local extinction caused by past climate change. However, our results for this species differ from simple expectations in several ways: (a) their absence at lower elevations is related to warm winter temperatures, not hot summer temperatures; (b) they appear to exclude a low‐elevation congener from higher elevations, not the converse; (c) they are apparently absent from many climatically suitable but low mountain ranges, seemingly “pushed off the top” by climates even warmer than those today; (d) despite the potential for dispersal among ranges during recent glacial periods (~18,000 years ago), populations in different ranges diverged ~4.5–0.5 million years ago and remained largely distinct; and (e) body temperatures are inversely related to climatic temperatures among sites. These results may have implications for many other Sky Island systems. More broadly, we suggest that Sky Island species may be relevant for predicting responses to future warming.     

Altered regulation of phosphatidylinositol 3-kinase signaling in cathepsin D-deficient brain

Cathepsin D (CD) is an essential lysosomal protease and mice lacking this enzyme exhibit neuropathology similar to that observed in brains of patients with neuronal ceroid lipofuscinosces (NCL/Batten disease), a group of autosomal recessive pediatric neurodegenerative diseases. CD-deficient (CD-/-) brains exhibit a dramatic induction of autophagic stress as defined by the aberrant accumulation of autophagosomes, which is concomitant with markers of apoptosis. However, the signaling abnormalities which lead to CD deficiency-induced neurodegeneration are poorly defined. Since phosphatidylinositol-3 kinase (PI3-K) is known to regulate both apoptosis and autophagy, PI3-K-mediated signaling events were assessed in CD-/- brain at P14 and P25-26. Compared to WT littermate controls, CD-/- cortical neurons exhibited a widespread decrease in phosphorylation of Akt (inactivation) and GSK3beta (disinhibition) at P25-26, while levels of total Akt and GSK3beta remained unchanged. This P25-26-specific decrease in phosphorylation of Akt and GSK-3beta in CD-/- brain coincided temporally with markers of apoptosis but followed the induction of autophagic stress observed at both P14 and P25-26. In addition, levels and/or activation of mTOR and Beclin were not affected by CD deficiency, suggesting that the accumulation of autophagosomes is not due to an increased synthesis of autophagosomes but rather from an inhibition of autophagosome recycling, due most likely to a compromise in lysosome function. Together these observations indicate a pronounced decrease in pro-survival PI3-K signaling in CD-/- brain that may contribute to autophagic stress-induced and apoptotic neuropathology.     

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion

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