Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.     

Integrated stream and wetland restoration: A watershed approach to improved water quality on the landscape

Water quality in Upper Sandy Creek, a headwater stream for the Cape Fear River in the North Carolina Piedmont, is impaired due to high N and P concentrations, sediment load, and coliform bacteria. The creek and floodplain ecosystem had become dysfunctional due to the effects of altered storm water delivery following urban watershed development where the impervious surface reached nearly 30% in some sub-watersheds. At Duke University, an 8-ha Stream and Wetland Assessment Management Park (SWAMP) was created in the lower portion of the watershed to assess the cumulative effect of restoring multiple portions of stream and former adjacent wetlands, with specific goals of quantifying water quality improvements. To accomplish these goals, a three-phase stream/riparian floodplain restoration (600 m), storm water reservoir/wetland complex (1.6 ha) along with a surface flow treatment wetland (0.5 ha) was ecologically designed to increase the stream wetland connection, and restore groundwater wetland hydrology. The multi-phased restoration of Sandy Creek and adjacent wetlands resulted in functioning riparian hydrology, which reduced downstream water pulses, nutrients, coliform bacteria, sediment, and stream erosion. Storm water event nutrient budgets indicated a substantial attenuation of N and P within the SWAMP project. Most notably, (NO₂⁻ + NO₃⁻)-N loads were reduced by 64% and P loads were reduced by 28%. Sediment retention in the stormwater reservoir and riparian wetlands showed accretion rates of 1.8 cm year⁻¹ and 1.1 cm year⁻¹, respectively. Sediment retention totaled nearly 500 MT year⁻¹.     

Characteristics of human dendritic cells generated in a microgravity analog culture system

Summary Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected, to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR⁺ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytoseAspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of Dc expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.     

Single molecule detection of DNA looping by NgoMIV restriction endonuclease

Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M, it was possible to detect and determine diffusion properties of looped DNA/protein complexes. FRET efficiency distributions revealed a subpopulation of complexes with an energy transfer efficiency of 30%, which appeared upon addition of enzyme in the picomolar to nanomolar concentration range (using 10(-11) M dsDNA). The concentration dependence, fluorescence burst size analysis, and fluorescence correlation analysis were all consistent with this subpopulation arising from a sequence specific interaction between an individual enzyme and a DNA molecule. A 30% FRET efficiency corresponds to a distance of approximately 65 A, which correlates well with the distance between the ends of the dsDNA molecule when bound to NgoMIV according to the crystal structure of this complex. Formation of the looped complexes was also evident in measurements of the diffusion times of freely diffusing DNA molecules with and without NgoMIV. At very high protein concentrations compared to the DNA concentration, FRET and fluorescence correlation spectroscopy results revealed the formation of larger DNA/protein complexes.     

Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.     

Resource assurance predicts specialist and generalist bee activity in drought

Many short-lived desert organisms remain in diapause during drought. Theoretically, the cues desert species use to continue diapause through drought should differ depending on the availability of critical resources, but the unpredictability and infrequent occurrence of climate extremes and reduced insect activity during such events make empirical tests of this prediction difficult. An intensive study of a diverse bee–plant community through a drought event found that bee specialists of a drought-sensitive host plant were absent in the drought year in contrast to generalist bees and to specialist bees of a drought-insensitive host plant. Different responses of bee species to drought indicate that the diapause cues used by bee species allow them to reliably predict host availability. Species composition of the bee community in drought shifted towards mostly generalist species. However, we predict that more frequent and extended drought, predicted by climate change models for southwest North America, will result in bee communities that are species-poor and dominated by specialist species, as found today in the most arid desert region of North America.     

Über somatische Induktionen auf die Keimdrüsen bei den Säugetieren

Ohne Zusammenfassung     

Pulse,Shunt and Storage: Hydrological Contraction Shapes Processing and Export of Particulate Organic Matter in River Networks

Ecosystems - Streams and rivers act as landscape-scale bioreactors processing large quantities of terrestrial particulate organic matter (POM). This function is linked to their flow regime, which...     

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.