Fluorescent substrates for ADAM15 useful for assaying and high throughput screening

A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs −2, –9, and −13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M⁻¹ s⁻¹ and 4300 M⁻¹ s⁻¹, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (k_cat/K_m) of 5200 M⁻¹ s⁻¹. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 μM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.     

Habitat selection by adult Golden Eagles Aquila chrysaetos during the breeding season and implications for wind farm establishment

Capsule: Global Positioning System (GPS)-tagged adult Golden Eagles Aquila chrysaetos breeding in forests in northern Sweden selected clear-cuts, coniferous forests with lichens and steep slopes during the breeding season but avoided wetlands and mixed forest.

Aims: To investigate the habitat selection patterns of tree-nesting Golden Eagles, and identify how potential conflicts with wind farm development could be minimized.

Methods: The study is based on GPS tracking data from 22 adult eagles. We estimated home range sizes using a biased random bridge approach and habitat selection patterns using resource selection functions following a use-availability design.

Results: Core home range size among adults was variable during the breeding season (5–30?km²). Individual movement extents were variable, but sexes did not significantly differ in their scale of movement. At the landscape scale, individuals selected for clear-cuts and coniferous forest with ground lichens, whereas wetland, water bodies and mixed forest were avoided. Steeper and south facing slopes were selected for, whereas, north facing slopes were avoided.

Conclusions: Potential conflicts between eagles and wind energy establishment can be reduced if wind farms are placed away from steep slopes, minimizing areas that are clear-cut during construction, and locating turbines within dense, young and other less favoured forest habitats.     

The structure of the littoral: effects of waterlily density and perch predation on sediment and plant‐associated macroinvertebrate communities

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Beef production potential of Norwegian Red and Holstein-Friesian bulls slaughtered at two ages

There is a paucity of data on the beef production potential of Norwegian Red (NOR) compared with 'modern' Holstein-Friesian (HF) cattle. The present study used a total of 64 bulls in a 2 × 2 factorial design study encompassing two breeds (HF and NOR) and two slaughter ages (485; E, and 610; L, days). The mean initial age and live weight of the HF bulls were 179 (s.d. 47.1) days and 203 (s.d. 64.0) kg, while the corresponding data for the NOR bulls were 176 (s.d. 39.7) days and 185 (s.d. 63.6) kg, respectively. Bulls were offered a 50 : 50 mixture (dry matter (DM) basis) of grass silage and concentrates. No breed × slaughter group interactions were recorded for any parameters evaluated (P > 0.05). HF bulls had higher (P < 0.001) DM intake and poorer (P < 0.01) efficiency of conversion of food to carcass gain than NOR bulls. HF bulls tended (P = 0.07) to have a higher rate of live-weight gain and were heavier (P < 0.001) at slaughter than NOR bulls, though both carcass weight and rate of carcass gain did not differ between the breeds (P > 0.05). NOR bulls had higher (P < 0.001) dressing proportion and carcass conformation score than HF bulls, while breed of bull had no influence (P > 0.05) on carcass fat classification, depth of subcutaneous fat, marbling score or on the weight of fat in the internal depots. Daily food intakes did not differ (P > 0.05) across the two slaughter age groups, though efficiency of conversion of food to carcass gain was poorer (P < 0.05) in the L compared with E bulls. Rate of live-weight gain was lower (P < 0.01) for L bulls, although rate of carcass gain did not differ (P > 0.05) between the E and L bulls. Increasing age at slaughter increased (P < 0.01 or greater) dressing proportion, carcass fat class, depth of subcutaneous fat, marbling score and internal fat depots, but had no effect (P > 0.05) on the carcass conformation score. Instrumental measures of meat quality indicated that meat from NOR bulls was tougher (P < 0.01) than meat from HF bulls, while delaying slaughter increased (P < 0.001) a* and C*ab, and decreased (P < 0.01) h0, indicating improved redness. It is concluded that NOR bulls have higher food efficiency and produce more highly conformed carcasses than HF bulls, but HF bulls produce more tender meat.     

Determination of the functional epitopes of human interleukin-18-binding protein by site-directed mutagenesis

The human interleukin (IL)-18-binding protein (hIL-18BP) is a naturally occurring antagonist of IL-18, a proinflammatory cytokine that is related to IL-1beta and has an important role in defense against microbial invaders. As its name implies, the hIL-18BP binds to IL-18 with high affinity and prevents the interaction of IL-18 with its receptor. We genetically modified the C terminus of hIL-18BP by appending a 15-amino acid biotinylation recognition site and a six-histidine tag and then performed site-directed mutagenesis to determine the functional epitopes that mediate efficient binding to IL-18. The mutated IL-18BPs were secreted from mammalian cells, captured by metal affinity chromatography, biotinylated in situ, eluted, and immobilized on streptavidin-coated chips. Using surface plasmon resonance, we identified seven amino acids of hIL-18BP which, when changed individually to alanine, caused an 8-750-fold decrease in binding affinity, largely because of increased off-rates. These seven amino acids localized to the predicted beta-strand c and d of hIL-18BP immunoglobulin-like domain, and most had hydrophobic side chains. Just two amino acids, tyrosine 97 and phenylalanine 104, contributed approximately 50% of the binding free energy. Information obtained from these studies could contribute to the design of molecular antagonists of IL-18 for treatment of inflammatory diseases.     

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.     

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.     

Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera

We produced an infectious vaccinia virus that expressed the B5R envelope glycoprotein fused to the enhanced green fluorescent protein (GFP), allowing us to visualize intracellular virus movement in real time. Previous transfection studies indicated that fusion of GFP to the C-terminal cytoplasmic domain of B5R did not interfere with Golgi localization of the viral protein. To determine whether B5R-GFP was fully functional, we started with a B5R deletion mutant that made small plaques and inserted the B5R-GFP gene into the original B5R locus. The recombinant virus made normal-sized plaques and acquired the ability to form actin tails, indicating reversal of the mutant phenotype. Moreover, immunogold electron microscopy revealed that both intracellular enveloped virions (IEV) and extracellular enveloped virions contained B5R-GFP. By confocal microscopy of live infected cells, we visualized individual fluorescent particles, corresponding to IEV in size and shape, moving from a juxtanuclear location to the periphery of the cell, where they usually collected prior to association with actin tails. The fluorescent particles could be seen emanating from cells at the tips of microvilli. Using a digital camera attached to an inverted fluorescence microscope, we acquired images at 1 frame/s. At this resolution, IEV movement appeared saltatory; in some frames there was no net movement, whereas in others movement exceeded 2 microm/s. Further studies indicated that IEV movement was reversibly arrested by the microtubule-depolymerizing drug nocodazole. This result, together with the direction, speed, and saltatory motion of IEV, was consistent with a role for microtubules in intracellular transport of IEV.     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.