Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.     

Binding and internalization of heparin by vascular smooth muscle cells

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.     

Mutational analysis of the histidine operon promoter of Salmonella typhimurium.

We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.     

Do Experience and Body Size Play a Role in Responses of Larval Ringed Salamanders,Ambystoma annulatum,to Predator Kairomones? Laboratory and Field Assays

Prey may experience ontogenetic changes in vulnerability to some predators, either because of changes in morphology or experience. If prey match their level of antipredator behavior to the level of predatory threat, prey responses to predators should reflect the appropriate level of threat for their stage of development. For larval salamanders, responses to predators may change with body size because larger larvae are less vulnerable to predation by gape‐limited predators or because fleeing responses by large salamanders may be more effective than for smaller salamanders. In a field experiment, small larval ringed salamanders, Ambystoma annulatum, responded to chemical stimuli (‘kairomones’) from predatory newts, Notophthalmus viridescens, with an antipredator response (decreased activity). Laboratory‐reared larvae decreased their activity following exposure to newt kairomones, indicating that larval ringed salamanders do not require experience with newts to recognize them as predators. In both experiments, larvae distinguished between chemical stimuli from newts and stimuli from tadpoles (non‐predators) and a blank control. In a third experiment, field‐caught (experienced) larvae showed a graded response to newt kairomones based on their body size: small larvae tended to decrease their activity while larger larvae showed no change or an increase in activity. This graded response was not observed for neutral stimuli, indicating that it is predator‐specific. Therefore, ringed salamander larvae exhibit threat‐sensitive ontogenetic changes in their response to chemical stimuli from predatory newts.     

The equilibrium constants and enthalpy changes for the esterification of glycine by ethanol in water and ethanol- water mixtures

Equilibrium constants, defined on the basis of the moles of all reactants and products including water, were found to be 4.6, 1.0 and 0.8 for the esterification of the glycinc cation at 20 degrees in a dilute aqueous sytem, and systems having 0.5 : 1 and 1 : 1 molar ratios of ethanol to water, respectively. When corrections were made for deviations from ideality a value of 5 +/- 1 was obtained for all three systems. Enthalpy changes were determined calorimetrically for the dilute aqueous and equimolar ethanol-water systems, and in each case was close to 1 kcal mole(-). The entropy change was calculated as 6 cal mole(-1) deg(-1).DeltaG(0') for the hydrolysis of the glycine ethyl ester cation at 20 degrees and pH 7 was calculated as -7.5 kcal mole(-1).     

Neurogranin Regulates Alcohol Sensitivity through AKT Pathway in the Nucleus Accumbens

Dysfunction of glutamate neurotransmission in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng) is exclusively expressed in the brain and mediates N‐methyl‐d ‐aspartate receptor (NMDAR) hypo‐function by regulating the intracellular calcium‐calmodulin (Ca²⁺‐CaM) pathway. Ng null mice (Ng^–/– mice) demonstrate increased alcohol drinking compared to wild‐type mice, while also showing less tolerance to the effect of alcohol. To identify the molecular mechanism related to alcohol seeking, both in vivo microdialysis and label‐free quantification proteomics comparing Ng genotype and effects of alcohol treatment on the NAc are utilized. There is significant difference in glutamate and gamma‐aminobutyric acid (GABA) neurotransmission between genotypes; however, alcohol administration normalizes both glutamate and GABA levels in the NAc. Using label‐free proteomics, 427 protein expression changes are identified against alcohol treatment in the NAc among 4347 total proteins detected. Bioinformatics analyses reveal significant molecular differences in Ng null mice in response to acute alcohol treatment. Ingenuity pathway analysis found that the AKT network is altered significantly between genotypes, which may increase the sensitivity of alcohol in Ng null mice. The pharmacoproteomics results presented here illustrate a possible molecular basis of the alcohol sensitivity through Ng signaling in the NAc.     

Increased high‐latitude photosynthetic carbon gain offset by respiration carbon loss during an anomalous warm winter to spring transition

Arctic and boreal ecosystems play an important role in the global carbon (C) budget, and whether they act as a future net C sink or source depends on climate and environmental change. Here, we used complementary in situ measurements, model simulations, and satellite observations to investigate the net carbon dioxide (CO₂) seasonal cycle and its climatic and environmental controls across Alaska and northwestern Canada during the anomalously warm winter to spring conditions of 2015 and 2016 (relative to 2010–2014). In the warm spring, we found that photosynthesis was enhanced more than respiration, leading to greater CO₂ uptake. However, photosynthetic enhancement from spring warming was partially offset by greater ecosystem respiration during the preceding anomalously warm winter, resulting in nearly neutral effects on the annual net CO₂ balance. Eddy covariance CO₂ flux measurements showed that air temperature has a primary influence on net CO₂ exchange in winter and spring, while soil moisture has a primary control on net CO₂ exchange in the fall. The net CO₂ exchange was generally more moisture limited in the boreal region than in the Arctic tundra. Our analysis indicates complex seasonal interactions of underlying C cycle processes in response to changing climate and hydrology that may not manifest in changes in net annual CO₂ exchange. Therefore, a better understanding of the seasonal response of C cycle processes may provide important insights for predicting future carbon–climate feedbacks and their consequences on atmospheric CO₂ dynamics in the northern high latitudes.     

Wildflower plantings promote blue orchard bee,Osmia lignaria (Hymenoptera: Megachilidae), reproduction in California almond orchards

Concerns over the availability of honeybees (Apis mellifera L.) to meet pollination demands have elicited interest in alternative pollinators to mitigate pressures on the commercial beekeeping industry. The blue orchard bee, Osmia lignaria (Say), is a commercially available native bee that can be employed as a copollinator with, or alternative pollinator to, honeybees in orchards. To date, their successful implementation in agriculture has been limited by poor recovery of bee progeny for use during the next spring. This lack of reproductive success may be tied to an inadequate diversity and abundance of alternative floral resources during the foraging period. Managed, supplementary wildflower plantings may promote O. lignaria reproduction in California almond orchards. Three wildflower plantings were installed and maintained along orchard edges to supplement bee forage. Plantings were seeded with native wildflower species that overlapped with and extended beyond almond bloom. We measured bee visitation to planted wildflowers, bee reproduction, and progeny outcomes across orchard blocks at variable distances from wildflower plantings during 2015 and 2016. Pollen provision composition was also determined to confirm O. lignaria wildflower pollen use. Osmia lignaria were frequently observed visiting wildflower plantings during, and after, almond bloom. Most O. lignaria nesting occurred at orchard edges. The greatest recovery of progeny occurred along the orchard edges having the closest proximity (80 m) to managed wildflower plantings versus edges farther away. After almond bloom, O. lignaria nesting closest to the wildflower plantings collected 72% of their pollen from Phacelia spp., which supplied 96% of the managed floral area. Phacelia spp. pollen collection declined with distance from the plantings, but still reached 17% 800 m into the orchard. This study highlights the importance of landscape context and proximity to supplementary floral resources in promoting the propagation of solitary bees as alternative managed pollinators in commercial agriculture.     

Aromatic interactions with naphthylalanine in a β‐hairpin peptide

Stable peptides have been explored as epitope mimics for protein–protein and protein–nucleic acid interactions; however, presentation of a regular structure is critical. Aromatic interactions are ubiquitous and are competent at stabilizing a β‐hairpin fold. The greatest stabilization has been reported from pairs of tryptophan side chains. Naphthylalanine residues are often used as tryptophan replacements, but it is not clear if 1‐naphthylalanine or 2‐naphthylalanine is adequate at replicating the geometry and stability observed with tryptophan aromatic interactions. Herein, a 12‐residue peptide has been constructed with laterally disposed aromatic amino acids. A direct comparison is made between tryptophan and other bicyclic, unnatural amino acids. Significant stabilization is gained from all bicyclic amino acids; however, geometric analysis shows that only 1‐naphthylalanine adopts a similar edge to face geometry as tryptophan, whereas the 2‐naphthylalanine appears most similar to a substituted phenylalanine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.