Roles of SIRT1 in the acute and restorative phases following induction of inflammation

Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents. Here, we show that in vivo endotoxin triggers a rapid and transient decline in ATP concentration in human peripheral blood leukocytes and murine peripheral blood leukocytes and liver, which is associated with a brief increase in expression of the autophagy indicator LC3-II. In both of these tissues, the ATP concentration reaches a nadir, and autophagy is induced between 2 and 4 h post-endotoxin infusion, and homeostasis is restored within 12 h. Mouse liver SIRT1 and AMP-activated protein kinase (AMPK) protein expression levels decline precipitously within 10 min and remain below detection levels for up to 12 h post-endotoxin administration. In marked contrast, the expression of HIF-1α is induced within 90 min and remains elevated for up to 12 h. The ATP recovery is delayed, and the increases in both HIF-1α expression and autophagy are prolonged in endotoxin-challenged SIRT1 liver knock-out mice. Resveratrol prevents the decline in ATP concentration and SIRT1 expression, as well as the increase in HIF-1α expression and autophagy in liver of endotoxin-challenged wild type mice but not in SIRT1 liver knock-out mice. These results provide novel insight into the state of both cellular bioenergetics and metabolic networks during the acute phase of systemic inflammation and suggest a role for SIRT1 in acute metabolic decline, as well as the restoration of metabolic homeostasis during an inflammatory challenge.     

Stream communities across a rural–urban landscape gradient

Rapid urbanization throughout the world is expected to cause extensive loss of biodiversity in the upcoming decades. Disturbances associated with urbanization frequently operate over multiple spatial scales such that local species extirpations have been attributed both to localized habitat degradation and to regional changes in land use. Urbanization also may shape stream communities by restricting species dispersal within and among stream reaches. In this patch-dynamics view, anthropogenic disturbances and isolation jointly reduce stream biodiversity in urbanizing landscapes. We evaluated predictions of stream invertebrate community composition and abundance based on variation in environmental conditions at five distinct spatial scales: stream habitats, reaches, riparian corridors and watersheds and their spatial location within the larger three-river basin. Despite strong associations between biodiversity loss and human density in this study, local stream habitat and stream reach conditions were poor predictors of community patterns. Instead, local community diversity and abundance were more accurately predicted by riparian vegetation and watershed landscape structure. Spatial coordinates associated with instream distances provided better predictions of stream communities than any of the environmental data sets. Together, results suggest that urbanization in the study region was associated with reduced stream invertebrate diversity through the alteration of landscape vegetation structure and patch connectivity. These findings suggest that maintaining and restoring watershed vegetation corridors in urban landscapes will aid efforts to conserve freshwater biodiversity.     

Characterization of dual agonists for kinin B1 and B2 receptors and their biased activation of B2 receptors

Kinin B1 and B2 receptors (kB1R and kB2R) play important roles in many physiological and pathological processes. In some cases, kB1R or kB2R activation can have overlapping or complementary beneficial effects, thus an activator of both receptors might be advantageous. We found that replacement of the C-terminal Arg in the natural kB2R activators bradykinin (BK) or kallidin (KD) with Lys (K(9)-BK or K(10)-KD) resulted in agonists that effectively stimulate the downstream signaling of both the kB1R and kB2R as measured by increased inositol turnover, intracellular calcium, ERK1/2 phosphorylation, arachidonic acid release and NO production. However, K(9)-BK and K(10)-KD displayed some characteristics of biased agonism for kB2Rs as indicated by the rapid kinetics of ERK1/2 phosphorylation induced by K(9)-BK or K(10)-KD compared with the prolonged response mediated by BK or KD. In contrast, kinetics of ERK phosphorylation stimulated by K(10)-KD activation of the kB1R was the same as that induced by known kB1R agonist des-Arg(10)-KD. Furthermore, the endocytosis of kB2Rs mediated by K(9)-BK and K(10)-KD was remarkably less than that induced by BK and KD respectively. K(10)-KD stimulated kB1R and kB2R-dependent calcium responses and ERK1/2 phosphorylation in bovine endothelial cells. In cytokine-treated human endothelial cells, K(10)-KD stimulated ERK1/2 phosphorylation and a transient peak of NO production that was primarily kB2R-dependent. K(10)-KD also stimulated prolonged NO production that was both kB1R and kB2R-dependent. These data provide the first examples of dual agonists of kB1R and kB2R, and a biased agonist of kB2R and may provide useful clues for developing dual modulators of kB1Rs and kB2Rs for potential therapeutic use.     

Microsatellite markers for the native Texas perennial grass, Panicum hallii (Poaceae)

? Premise of the study: We developed microsatellites for Panicum hallii for studies of gene flow, population structure, breeding experiments, and genetic mapping. ? Methods and Results: Next-generation (454) genomic sequence data were used to design markers. Eighteen robust markers were discovered, 15 of which were polymorphic across six accessions of P. hallii var. hallii. Fourteen of the markers cross-amplified in a P. capillare accession. For the 15 polymorphic markers, the total number of alleles per locus ranged from two to 26 (mean: 11.0) across six populations (11-19 individuals per population). Observed heterozygosity (mean: 0.031) was 13.7 times lower than the expected heterozygosity (mean: 0.426). ? Conclusions: The deficit of heterozygous individuals is consistent with P. hallii having a high rate of self-fertilization. These markers will be useful for studies in P. hallii and related species.     

Mapping of ionomic traits in Mimulus guttatus reveals Mo and Cd QTLs that colocalize with MOT1 homologues

Natural variation in the regulation of the accumulation of mineral nutrients and trace elements in plant tissues is crucial to plant metabolism, development, and survival across different habitats. Studies of the genetic basis of natural variation in nutrient metabolism have been facilitated by the development of ionomics. Ionomics is a functional genomic approach for the identification of the genes and gene networks that regulate the elemental composition, or ionome, of an organism. In this study, we evaluated the genetic basis of divergence in elemental composition between an inland annual and a coastal perennial accession of Mimulus guttatus using a recombinant inbred line (RIL) mapping population. Out of 20 elements evaluated, Mo and Cd were the most divergent in accumulation between the two accessions and were highly genetically correlated in the RILs across two replicated experiments. We discovered two major quantitative trait loci (QTL) for Mo accumulation, the largest of which consistently colocalized with a QTL for Cd accumulation. Interestingly, both Mo QTLs also colocalized with the two M. guttatus homologues of MOT1, the only known plant transporter to be involved in natural variation in molybdate uptake.     

How to build a mammalian super-predator

Questions surrounding the biology of large fossil predators that differ markedly from living forms have long intrigued palaeobiologists. Among such taxa few have excited more interest than sabertooth cats, whose distinctive hypertrophied canines are suggestive of killing behaviors and feeding ecologies that may have departed widely from those of extant carnivores. Moreover, considerable variation among sabertooth species is further suggestive of intriguing differences within the group. Behavior and ecology in another large, extinct mammalian carnivore, the Australian marsupial lion (Thylacoleo carnifex), has also proven contentious. In this study, we assemble a wide range of cranio-dental and postcranial indices in a dataset including machairodont sabertooths, T. carnifex and an extensive sample of extant taxa in order to examine the palaeobiology of these charismatic fossil carnivores. Results of multivariate analyses point to significant relationships between behavior and overall body proportions in extant mammalian carnivores. Postcranial morphologies of two American dirk-tooth species of sabertooth (Smilodon) depart greatly from those of living felids and group most closely with bears among living placentals. Scimitar-tooth species of Homotherium and Machairodus cluster with modern pantherine cats. The marsupial lion groups with Smilodon. If these latter two phylogenetically disparate clades do represent a specialized, robust ecomorph adapted to predation on large prey, then it is a body plan that might be effectively identified on the basis of a handful of ‘bear-like’ postcranial features in combination with a more typically ‘felid-like’ carnassialization of the cheektooth row.     

Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous “treatment–control” experimental design was performed to detect K⁺-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC–EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment–control experiments performed on the same initial PC12 cell population.     

G Protein beta gamma subunits act on the catalytic domain to stimulate Bruton's agammaglobulinemia tyrosine kinase.

G proteins are critical cellular signal transducers for a variety of cell surface receptors. Both alpha and betagamma subunits of G proteins are able to transduce receptor signals. Several direct effect molecules for Gbetagamma subunits have been reported; yet the biochemical mechanism by which Gbetagamma executes its modulatory role is not well understood. We have shown that Gbetagamma could directly increase the kinase activity of Bruton's tyrosine kinase (Btk) whose defects are responsible for X chromosome-linked agammaglobulinemia in patients. The well characterized interaction of Gbetagamma with the PH (pleckstrin homology)/TH (Tec-homology) module of Btk was proposed to be the underlying activation mechanism. Here we show that Gbetagamma also interacts with the catalytic domain of Btk leading to increased kinase activity. Furthermore, we showed that the PH/TH module is required for Gbetagamma-induced membrane translocation of Btk. The membrane anchorage is also dependent on the interaction of Btk with phosphatidylinositol 3,4,5-trisphosphate, the product of phosphoinositide 3-kinase. These data support a dual role for Gbetagamma in the activation of Btk signaling function, namely membrane translocation and direct regulation of Btk catalytic activity.