Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles.     

Hepatic metabolism of vasoactive intestinal polypeptide (VIP) in the rat

Vasoactive intestinal polypeptide (VIP) is released into the portal circulation by a meal stimulus, but is rapidly cleared from plasma. Although it is known to bind to receptors on liver cells, the role of the liver in the clearance of VIP is not clearly defined. We therefore studied the disappearance of VIP in recirculating and in single pass isolated perfused rat liver (IPRL) preparations. Disappearance of added VIP was rapid in recirculating IPRL experiments with a half life of ca. 30 min. In single-pass steady-state studies in which livers were perfused at 16 ml/min for 30 min, clearance of VIP was complete (16 ml/min) at concentrations of 500 fmol/ml, but clearance fell to 3 and 1 ml/min at perfusate concentrations of 8 and 40 pmol/ml respectively. Further experiments to evaluate whether VIP was disappearing in perfusate itself demonstrated substantial metabolism of VIP in perfusate which had previously been circulated through a liver for 90 min. The products of metabolism were identical to those found in the IPRL. We conclude that VIP is rapidly cleared as it passes through the isolated perfused rat liver model with a significant proportion of clearance attributable to release of a peptidase from the liver into the perfusate.     

How Self-Generated Thought Shapes Mood—The Relation between Mind-Wandering and Mood Depends on the Socio-Temporal Content of Thoughts

Recent work has highlighted that the generation of thoughts unrelated to the current environment may be both a cause and a consequence of unhappiness. The current study used lag analysis to examine whether the relationship between self-generated thought and negative affect depends on the content of the thoughts themselves. We found that the emotional content could strongly predict subsequent mood (e.g. negative thoughts were associated with subsequent negative mood). However, this direct relationship was modulated by the socio-temporal content of the thoughts: thoughts that were past- and other-related were associated with subsequent negative mood, even if current thought content was positive. By contrast, future- and self-related thoughts preceded improvements of mood, even when current thought content was negative. These results highlight the important link between self-generated thought and mood and suggest that the socio-temporal content plays an important role in determining whether an individual''s future affective state will be happy or sad.     

Identification of Astrotactin2 as a Genetic Modifier That Regulates the Global Orientation of Mammalian Hair Follicles

Planar cell polarity (PCP) signaling controls the global orientation of surface structures, such as hairs and bristles, in both vertebrates and invertebrates. In Frizzled6 ^-/- (Fz6 ^-/-) mice, hair follicle orientations on the head and back are nearly random at birth, but reorient during early postnatal development to eventually generate a nearly parallel anterior-to-posterior array. We report the identification of a naturally occurring exon 5 deletion in Astrotactin2 (Astn2) that acts as a recessive genetic modifier of the Fz6 ^-/- hair patterning phenotype. A genetically engineered Astn2 exon 5 deletion recapitulates the modifier phenotype. In Fz6 ^-/- ;Astn2 ^ex5del/del mice, hair orientation on the back is subtly biased from posterior-to-anterior, leading to a 180-degree orientation reversal in mature mice. These experiments suggest that Astn2, an endosomal membrane protein, modulates PCP signaling.     

Paleoindian Unifacial Stone Tool ‘Spurs’: Intended Accessories or Incidental Accidents?

Paleoindian unifacial stone tools frequently exhibit distinct, sharp projections, known as “spurs”. During the last two decades, a theoretically and empirically informed interpretation–based on individual artifact analysis, use-wear, tool-production techniques, and studies of resharpening–suggested that spurs were sometimes created intentionally via retouch, and other times created incidentally via resharpening or knapping accidents. However, more recently Weedman strongly criticized the inference that Paleoindian spurs were ever intentionally produced or served a functional purpose, and asserted that ethnographic research “demonstrates that the presence of so called ‘graver’ spurs does not have a functional significance.” While ethnographic data cannot serve as a direct test of the archaeological record, we used Weedman’s ethnographic observations to create two quantitative predictions of the Paleoindian archaeological record in order to directly examine the hypothesis that Paleoindian spurs were predominantly accidents occurring incidentally via resharpening and reshaping. The first prediction is that the frequency of spurs should increase as tool reduction proceeds. The second prediction is that the frequency of spurs should increase as tool breakage increases. An examination of 563 unbroken tools and 629 tool fragments from the Clovis archaeological record of the North American Lower Great Lakes region showed that neither prediction was consistent with the notion that spurs were predominately accidents. Instead, our results support the prevailing viewpoint that spurs were sometimes created intentionally via retouch, and other times, created incidentally via resharpening or knapping accidents. Behaviorally, this result is consistent with the notion that unifacial stone tools were multifunctional implements that enhanced the mobile lifestyle of Pleistocene hunter-gatherers.     

Routes to improving the reliability of low level DNA analysis using real-time PCR

Background  

Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated.     

Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart.

We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.