Structures of the cancer-related Aurora-A,FAK, and EphA2 protein kinases from nanovolume crystallography

Protein kinases are important drug targets in human cancers, inflammation, and metabolic diseases. This report presents the structures of kinase domains for three cancer-associated protein kinases: ephrin receptor A2 (EphA2), focal adhesion kinase (FAK), and Aurora-A. The expression profiles of EphA2, FAK, and Aurora-A in carcinomas suggest that inhibitors of these kinases may have inherent potential as therapeutic agents. The structures were determined from crystals grown in nanovolume droplets, which produced high-resolution diffraction data at 1.7, 1.9, and 2.3 A for FAK, Aurora-A, and EphA2, respectively. The FAK and Aurora-A structures are the first determined within two unique subfamilies of human kinases, and all three structures provide new insights into kinase regulation and the design of selective inhibitors.     

Pea aphid dropping behavior diminishes foraging efficiency of a predatory ladybeetle

Antipredator defensive behaviors are a well‐studied and often crucial part of prey life histories, but little has been done to quantify how such behaviors affect natural enemies, their foraging, and their effectiveness as biological control agents. We explored how the generalist predatory coccinellid Harmonia axyridis Pallas (Coleoptera: Coccinellidae) affects the dropping behavior of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), and in turn, how that defensive behavior affects the foraging efficiency of the predator. Experimental arenas that allowed or prevented pea aphid dropping were compared to determine how dropping influences the foraging of multiple life stages of H. axyridis: second instars, fourth instars, and adults. Dropping reduced predation on aphids by all ladybeetle life stages. Despite older predators inducing more dropping, aphid dropping reduced predation by approximately 40% across all ladybeetle life stages. Aphid dropping and predator consumption of aphids were both correlated with how much the predator moved, which also increased with predator life stage. We suggest that the high rates of dropping induced by H. axyridis and the subsequent decrease in H. axyridis foraging efficiency may partially explain why H. axyridis is less effective at controlling pea aphids than it is at controlling other aphid species that do not drop.     

Pax7 shows higher satellite cell frequencies and concentrations within intrafusal fibers of muscle spindles

Intrafusal fibers within muscle spindles make up a small subpopulation of muscle fibers. These proprioceptive fibers differ from most extrafusal fibers because, even in maturity, their diameters remain small, and they retain expression of developmental myosins. Although both extrafusal and intrafusal fibers contain satellite cells (SCs), comparatively little is known about intrafusal SCs. Analyzing chicken fast-phasic posterior (PLD) and slow-tonic anterior (ALD) latissimus dorsi muscles, we show that SCs of both intrafusal and extrafusal fibers express Pax7. We further test the hypotheses that intrafusal fibers display parameters reflective of extrafusal immaturity. These hypotheses are that intrafusal fibers contain (a) higher SC frequencies (number of SC nuclei/all nuclei within basal lamina) and concentrations (closer together) and (b) smaller myonuclear domains than do adjacent extrafusal fibers. IHC techniques were applied to PLD and ALD muscles excised at 30 and 138 days posthatch. The hypotheses were validated, suggesting that intrafusal fibers have greater capacities for growth, regeneration, and repair than do adjacent extrafusal fibers. During maturation, extrafusal and intrafusal fibers show similar trends of decreasing SC frequencies and concentrations and increases in myonuclear domains. Thus, extrafusal and intrafusal fibers alike should exhibit reduced capacities for growth, regeneration, and repair during maturation.     

A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.     

Rapid acid treatment of <Emphasis Type="Italic">Escherichia coli</Emphasis>: transcriptomic response and recovery

Background  

Many E. coli genes show pH-dependent expression during logarithmic growth in acid (pH 5–6) or in base (pH 8–9). The effect of rapid pH change, however, has rarely been tested. Rapid acid treatment could distinguish between genes responding to external pH, and genes responding to cytoplasmic acidification, which occurs transiently following rapid external acidification. It could reveal previously unknown acid-stress genes whose effects are transient, as well as show which acid-stress genes have a delayed response.     

Fluid pressures at the shoe–floor–contaminant interface during slips: Effects of tread & implications on slip severity

Previous research on slip and fall accidents has suggested that pressurized fluid between the shoe and floor is responsible for initiating slips yet this effect has not been verified experimentally. This study aimed to (1) measure hydrodynamic pressures during slipping for treaded and untreaded conditions; (2) determine the effects of fluid pressure on slip severity; and (3) quantify how fluid pressures vary with instantaneous resultant slipping speed, position on the shoe surface, and throughout the progression of the slip. Eighteen subjects walked on known dry and unexpected slippery floors, while wearing treaded and untreaded shoes. Fluid pressure sensors, embedded in the floor, recorded hydrodynamic pressures during slipping. The maximum fluid pressures (mean+/−standard deviation) were significantly higher for the untreaded conditions (124+/−75 kPa) than the treaded conditions (1.1+/−0.29 kPa). Maximum fluid pressures were positively correlated with peak slipping speed (r=0.87), suggesting that higher fluid pressures, which are associated with untreaded conditions, resulted in more severe slips. Instantaneous resultant slipping speed and position of sensor relative to the shoe sole and walking direction explained 41% of the fluid pressure variability. Fluid pressures were primarily observed for untreaded conditions. This study confirms that fluid pressures are relevant to slipping events, consistent with fluid dynamics theory (i.e. the Reynolds equation), and can be modified with shoe tread design. The results suggest that the occurrence and severity of unexpected slips can be reduced by designing shoes/floors that reduce underfoot fluid pressures.     

A comparison of methods for the measurement of protein turnover in vivo.

Steady-state rates of turnover of two single proteins were measured in vivo by two independent methods. The fractional rate of synthesis of liver ornithine aminotransferase, measured by a continuous infusion of L-[2,6-3H]tyrosine, was 0.42 day-1, whereas in the same animals the fractional rate of degradation measured by loss of radioactivity from amino acids labelled via [14C]bicarbonate was 0.40 day-1. The agreement between methods confirms the reliability of each method for the study of hepatic protein turnover. In contrast, [14C]bicarbonate-labelled amino acids are extensively reutilized in muscle, and are therefore unsuitable for measuring rates of muscle protein breakdown.