Dynamic assembly of the urokinase-type plasminogen activator signaling receptor complex determines the mitogenic activity of urokinase-type plasminogen activator

The urokinase-type plasminogen activator (uPA) receptor (uPAR) functions in concert with co-receptors, including integrins, FPR-like receptor-1/lipoxin A4 receptor, and the epidermal growth factor receptor (EGFR), to initiate cell signaling. uPAR co-receptors may be dynamically organized into a multiprotein signaling receptor complex. In Chinese hamster ovary-K1 (CHO-K1) cells, uPA-binding to uPAR activates ERK/MAP kinase, even though these cells do not express the EGFR; however, when CHO-K1 cells are transfected to express the EGFR, ERK activation becomes EGFR-dependent. In this study, we demonstrate that ERK activation in response to uPA follows equivalent biphasic kinetics in EGFR-expressing and -deficient CHO-K1 cells. In both cell types, the response is pertussis toxin-sensitive; however, uPA promotes cell proliferation exclusively in the EGFR-expressing cells. uPA-induced mitogenic activity requires activation of both STAT5b and ERK. STAT5b was tyrosine-phosphorylated, in response to uPA, only in EGFR-expressing cells. uPA-induced cell proliferation was blocked by dominant-negative MEK1, dominant-negative STAT5b, and by expression of an EGFR that is mutated at Tyr-845, which is essential for STAT5b activation. In two cell culture models of uPA-stimulated breast cancer growth, MDA-MB 468 cells treated with uPA and MCF-7 cells treated with uPA-plasminogen activator inhibitor-1 complex, proliferation was completely inhibited when EGFR expression or activity was blocked. We conclude that expression and assembly of uPAR co-receptors in a specific cell type determines the response to uPA. The EGFR selectively cooperates with uPAR to mediate mitogenesis.     

Anaerobic Biodegradation of Alkylbenzenes in Laboratory Microcosms Representing Ambient Conditions

A microcosm study was performed to document the anaerobic biodegradation of benzene, toluene, ethylbenzene, m- xylene, and/or o-xylene in petroleum-contaminated aquifer sediment from sites in Michigan (MI) and North Carolina (NC) and relate the results to previous field investigations of intrinsic bioremediation. Laboratory microcosms, designed to simulate ambient conditions, were constructed under anaerobic conditions with sediment and groundwater from source, mid-plume, and end-plume locations at each site. The general patterns of biodegradation and electron acceptor utilization in the microcosms were consistent with field data. At the MI site, methane was produced after a moderate lag period, followed by toluene degradation in all sets of microcosms. At the NC site, biodegradation of the target compounds was not evident in the source area microcosms. In the mid-plume microcosms, toluene and o-xylene biodegraded first, followed by m-xylene and benzene, a pattern consistent with contaminant decay along the plume length. Chemical extraction of microcosm sediment at the beginning and end of me incubation indicated that iron-reducing conditions were dominant and iron reduction occurred on a sediment fraction not extracted by 0.5N HC1. In the end-plume microcosms, degradation of benzene, toluene, and xylene isomers occurred but was variable between replicates. Consistent with field data, dissolved concentrations of the target contaminant(s) persisted at low but detectable levels (0.05 to 0.25 μM) in microcosms from both sites where biodegradation was measured.     

Sensory Aspects of Trail-Following Behaviors in the Asian Longhorned Beetle, <Emphasis Type="Italic">Anoplophora glabripennis</Emphasis>

Anoplophora glabripennis has a complex suite of mate-finding behaviors, the functions of which are not entirely understood. These behaviors are elicited by a number of factors, including visual and chemical cues. Chemical cues include a male-produced volatile semiochemical acting as a long-range sex pheromone, a female-produced cuticular hydrocarbon blend serving as a sex-identification contact pheromone, and a recently identified female-produced trail sex pheromone that is followed by mate-seeking males. However, the sensory appendages and sensilla on these appendages used to detect the trail sex pheromone are unknown. We evaluated the ability of virgin male A. glabripennis to follow a sex pheromone trail after removal of the terminal four antennal segments and/or the maxillary and labial palps using a two-choice behavioral bioassay. We also tested the ability of males to follow the trail sex pheromone using volatile pheromone cues only, without physical contact with the pheromone. Results indicate that the palps are primarily responsible for sensing the pheromone, with males lacking palps unable to respond behaviorally to the trail sex pheromone. Under the conditions of this study, males could not follow the sex pheromone trail without direct contact, suggesting that olfaction may not be involved in detection of this pheromone. However, we did not determine to what degree the trail pheromone chemicals can volatilize under our experimental conditions. This work is important in elucidating the behaviors and sensory structures involved in mate-finding by this species on host trees, and these studies may help determine whether the trail sex pheromone has applications for monitoring and management.     

Intrinsic Immunity Shapes Viral Resistance of Stem Cells

1. Download high-res image (223KB)
2. Download full-size image

     

Thermostable single domain antibody–maltose binding protein fusion for Bacillus anthracis spore protein BclA detection

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb–A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K_D ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP–sdAb gave a yield of approximately 100 mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb–A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1 mg/ml for 1 h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1 h at 90 °C. The PfuMBP–sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb–A5 both as a capture reagent and as a detection reagent.     

Targeted sugar provision promotes parasitism of the cereal leaf beetle Oulema melanopus

• 1 Parasitoids may often lack access to sugar (e.g. floral nectar) in agricultural settings. Strategically timed spraying of host plants with sugar solution may provide one means of enhancing parasitism at the same time as minimizing nontarget effects (e.g. benefiting the pest itself).
• 2 Sucrose was sprayed in wheat fields of northern Utah (U.S.A.) to assess the effects on parasitism of the cereal leaf beetle Oulema melanopus by the larval parasitoid Tetrastichus julis.
• 3 Early‐season sugar provisioning, when larvae of the pest were first hatching and parasitoid adults were newly emerged, did not affect the numbers of cereal leaf beetle larvae that matured in treated plots but increased parasitism rates of beetle larvae by four‐fold in 2006 and by seven‐fold in 2007.
• 4 No net influx of adult parasitoids into plots was detected after the application of sugar. Locally‐emerging parasitoids may have spent less time searching for their own food needs versus hosts. A laboratory experiment also confirmed that access to sucrose significantly increased parasitoid longevity.
• 5 The field experimental results obtained demonstrate that applications of sugar, implemented to target a key time of the growing season when benefits are maximized for parasitoids and minimized for their hosts, can strongly promote parasitism of the cereal leaf beetle in wheat fields.

     

Effects of temperature on Anoplophora glabripennis (Coleoptera: Cerambycidae) larvae and pupae

Developmental thresholds, degree-days for development, larval weights, and head capsule widths for each larval instar and the pupal stage of Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) were studied at eight constant temperatures (5, 10, 15, 20, 25, 30, 35, and 40°C) for two source populations (Ravenswood, Chicago, IL [IL], and Bayside, Queens, NY [NY]). The estimated lower threshold temperature for development of instars 1-5 and the pupal stage was near 10°C and was near 12°C for the higher instars. Developmental rate was less temperature sensitive for instars 5-9 compared with instars 1-4. Development for all but the first instar was inhibited at constant temperatures >30°C, and all instars failed to develop at 40°C. Although the two source populations had similar responses to temperature, IL larvae were heavier than those from NY. Temperature and its influence on larval weight had profound impacts on whether a larva proceeded to pupation. Based on the temperature effects detailed here, larval development and pupation should be possible in most of the continental United States where suitable hosts are available. These data can be used to develop a degree-day model to estimate beetle phenology; however, at least 2°C should be added to air temperatures to adjust for the mediation of temperature by the wood. These data provide a basis for predicting the potential geographical range of this species and for developing phenological models to predict the timing of immature stages, both of which are important for management programs.