Dendrobatid poison frogs sequester lipophilic alkaloids from their arthropod prey to use as a form of chemical defense. Some dendrobatid frogs seasonally migrate between the leaf litter of the forest floor in the dry season to the canopy in the wet season, which may yield differences in prey (arthropods) and therefore alkaloid availability over space and time. Here, we document a seasonal vertical migration of Andinobates fulguritus (the yellow‐bellied poison frog) from ground to canopy between dry and wet seasons. We observed turnover in alkaloid composition between seasons and found that dry season frogs contained a lower relative quantity of alkaloids; however, there was no change in alkaloid richness between seasons. The 77 alkaloids of 13 structural classes identified in this population appear to be derived mostly from mites and ants, though the two most common alkaloids were mite derived. Our observed shifts in defensive profiles are consistent with well‐documented turnover in mite and ant communities between seasons and vertical strata. As climate change is expected to lengthen and strengthen dry seasons in many tropical regions, our results suggest that arboreal poison frogs forced to the ground for longer periods of time may see a shift in the abundance of alkaloids, possibly decreasing their defensive potential. This study provides further predictions for the wide‐reaching effects of climate change, even as nuanced as charismatic poison frogs losing their poisons. Abstract in Spanish is available with online material. 相似文献
The River Continuum Concept implies that consumers in headwater streams have greater dietary access to terrestrial basal resources, but recent studies have highlighted the dietary importance of high-quality algae. Algae provide consumers with physiologically important omega-3 (n-3) polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). However, terrestrial plants and most benthic stream algae lack the long-chain (LC) n-3 PUFA docosahexaenoic acid (DHA, 22:6n-3), which is essential for neural development in fish and other vertebrates. We sampled subalpine streams to investigate how the PUFA composition of neural (brain and eyes), muscle, and liver tissues of freshwater fish is related to their potential diets (macroinvertebrates, epilithon, fresh and conditioned terrestrial leaves). The PUFA composition of consumers was more similar to epilithon than to terrestrial leaves. Storage lipids of eyes most closely resembled dietary PUFA (aquatic invertebrates and algae). However, DHA and arachidonic acid (ARA, 20:4n-6) were not directly available in the diet but abundant in organs. This implies that algal PUFA were selectively retained or were produced internally via enzymatic PUFA conversion by aquatic consumers. This field study demonstrates the nutritional importance of algal PUFA for neural organs in aquatic consumers of headwater regions.
Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure. 相似文献
Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin''s Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery. 相似文献
There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity. 相似文献
Hydrophobins represent a class of unique fungal proteins that have low molecular mass, are cysteine rich, and can self-assemble
into two-dimensional arrays at water/air interfaces. These highly surface-active proteins are able to decrease the surface
tension of water, thus allowing fungal structures to penetrate hydrophobic–hydrophilic barriers. Due to their unusual biophysical
properties, hydrophobins have been suggested for use in a wide range of biotechnological applications. Here we describe a
simple method for producing a functionally active class I hydrophobin derived from the entomopathogenic fungus, Beauveria bassiana, in an E. coli host. N-terminal modifications were required for proper expression and purification, and the hydrophobin was expressed as
a fusion partner to a cleavable N-terminus chitin-binding domain-intein construct. The protein was purified and reconstituted
from E. coli inclusion bodies. Self-assembly of the recombinant hydrophobin was followed kinetically using a thioflavin T fluorescence
binding assay, and contact angle measurements of purified recombinant hydrophobin protein (mHyd2) films on a variety of substrata
demonstrated its surface modification ability, which remained stable for at least 4 months. Filament or fibril-like structures
were imaged using atomic force and transmission electron microscopy. These data confirmed the functional properties of the
purified protein and indicate amino acid flexibility at the N-terminus, which can be exploited for various applications of
these proteins. 相似文献
Traffic through late endolysosomal compartments is regulated by sequential signaling of small G proteins of the Rab5 and Rab7 families. The Saccharomyces cerevisiae Vps-C protein complexes CORVET (class C core vacuole/endosome tethering complex) and HOPS (homotypic fusion and protein transport) interact with endolysosomal Rabs to coordinate their signaling activities. To better understand these large and intricate complexes, we performed interaction surveys to assemble domain-level interaction topologies for the eight Vps-C subunits. We identified numerous intersubunit interactions and up to six Rab-binding sites. Functional modules coordinate the major Rab interactions within CORVET and HOPS. The CORVET-specific subunits, Vps3 and Vps8, form a subcomplex and physically and genetically interact with the Rab5 orthologue Vps21. The HOPS-specific subunits, Vps39 and Vps41, also form a subcomplex. Both subunits bind the Rab7 orthologue Ypt7, but with distinct nucleotide specificities. The in vivo functions of four RING-like domains within Vps-C subunits were analyzed and shown to have distinct functions in endolysosomal transport. Finally, we show that the CORVET- and HOPS-specific subunits Vps3 and Vps39 bind the Vps-C core through a common region within the Vps11 C-terminal domain (CTD). Biochemical and genetic experiments demonstrate the importance of these regions, revealing the Vps11 CTD as a key integrator of Vps-C complex assembly, Rab signaling, and endosomal and lysosomal traffic. 相似文献
Development and preclinical testing of new cancer therapies is limited by the scarcity of in vivo models that authentically reproduce tumor growth and metastatic progression. We report new models for breast tumor growth and metastasis in the form of transplantable tumors derived directly from individuals undergoing treatment for breast cancer. These tumor grafts illustrate the diversity of human breast cancer and maintain essential features of the original tumors, including metastasis to specific sites. Co-engraftment of primary human mesenchymal stem cells maintains phenotypic stability of the grafts and increases tumor growth by promoting angiogenesis. We also report that tumor engraftment is a prognostic indicator of disease outcome for women with newly diagnosed breast cancer; orthotopic breast tumor grafting is a step toward individualized models for tumor growth, metastasis and prognosis. This bank of tumor grafts also serves as a publicly available resource for new models in which to study the biology of breast cancer. 相似文献
Fluticasone furoate (FF) is a novel long-acting inhaled corticosteroid (ICS). This double-blind, placebo-controlled randomized study evaluated the efficacy and safety of FF 200 mcg or 400 mcg once daily, either in the morning or in the evening, and FF 200 mcg twice daily (morning and evening), for 8 weeks in patients with persistent asthma.
Methods
Asthma patients maintained on ICS for ≥ 3 months with baseline morning forced expiratory volume in one second (FEV1) 50-80% of predicted normal value and FEV1 reversibility of ≥ 12% and ≥ 200 ml were eligible. The primary endpoint was mean change from baseline FEV1 at week 8 in pre-dose (morning or evening [depending on regimen], pre-rescue bronchodilator) FEV1.
Results
A total of 545 patients received one of five FF treatment groups and 101 patients received placebo (intent-to-treat population). Each of the five FF treatment groups produced a statistically significant improvement in pre-dose FEV1 compared with placebo (p < 0.05). FF 400 mcg once daily in the evening and FF 200 mcg twice daily produced similar placebo-adjusted improvements in evening pre-dose FEV1 at week 8 (240 ml vs. 235 ml). FF 400 mcg once daily in the morning, although effective, resulted in a smaller improvement in morning pre-dose FEV1 than FF 200 mcg twice daily at week 8 (315 ml vs. 202 ml). The incidence of oral candidiasis was low (0-4%) and UC excretion was comparable with placebo for all FF groups.
Conclusions
FF at total daily doses of 200 mcg or 400 mcg was significantly more effective than placebo. FF 400 mcg once daily in the evening had similar efficacy to FF 200 mcg twice daily and all FF regimens had a safety tolerability profile generally similar to placebo. This indicates that inhaled FF is an effective and well tolerated once-daily treatment for mild-to-moderate asthma.