Trade-offs between cost of ingestion and rate of intake drive defensive toxin use.

Animals that ingest toxins can become unpalatable and even toxic to predators and parasites through toxin sequestration. Because most animals rapidly eliminate toxins to survive their ingestion, it is unclear how populations transition from susceptibility and toxin elimination to tolerance and accumulation as chemical defence emerges. Studies of chemical defence have generally focused on species with active toxin sequestration and target-site insensitivity mutations or toxin-binding proteins that permit survival without necessitating toxin elimination. Here, we investigate whether animals that presumably rely on toxin elimination for survival can use ingested toxins for defence. We use the A4 and A3 Drosophila melanogaster fly strains from the Drosophila Synthetic Population Resource (DSPR), which respectively possess high and low metabolic nicotine resistance among DSPR fly lines. We find that ingesting nicotine increased A4 but not A3 fly survival against Leptopilina heterotoma wasp parasitism. Further, we find that despite possessing genetic variants that enhance toxin elimination, A4 flies accrued more nicotine than A3 individuals, likely by consuming more medium. Our results suggest that enhanced toxin metabolism can allow greater toxin intake by offsetting the cost of toxin ingestion. Passive toxin accumulation that accompanies increased toxin intake may underlie the early origins of chemical defence.

Correction to: Repository corticotropin injection versus corticosteroids for protection against renal damage in a focal segmental glomerulosclerosis rodent model.

An amendment to this paper has been published and can be accessed via the original article.

Repository corticotropin injection versus corticosteroids for protection against renal damage in a focal segmental glomerulosclerosis rodent model.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) causes renal fibrosis and may lead to kidney failure. FSGS and its common complication, proteinuria, are challenging to treat. Corticosteroids are ineffective in many patients with FSGS, and alternative treatments often yield suboptimal responses. Repository corticotropin injection (RCI; Acthar® Gel), a naturally sourced complex mixture of purified adrenocorticotropic hormone analogs and other pituitary peptides, may have beneficial effects on idiopathic FSGS via melanocortin receptor activation. METHODS: Two studies in a preclinical (female Sprague-Dawley rats) puromycin aminonucleoside FSGS model assessed the effect of RCI on renal function and morphology: an 8-week comparison of a single RCI dose with methylprednisolone (N = 27), and a 12-week chronic RCI dose range study (N = 34). Primary outcomes were proteinuria and renal pathology improvements for measures of renal fibrosis, tubular damage, glomerular injury, and total kidney injury score. Impact of RCI treatment was also determined by assessing urinary biomarkers for renal injury, podocyte expression of podoplanin (a biomarker for injury), podocyte effacement by electron microscopy, and histological staining for fibrosis biomarkers. RESULTS: Compared with saline treatment, RCI 30 IU/kg significantly reduced proteinuria, with a 38% reduction in peak mean urine protein levels on day 28 in the 8-week model, and RCI 10 IU/kg, 30 IU/kg, and 60 IU/kg reduced peak mean urine protein in the 12-week model by 18, 47, and 44%, respectively. RCI also showed significant dose-dependent improvements in fibrosis, interstitial inflammation, tubular injury, and glomerular changes. Total kidney injury score (calculated from histopathological evaluations) demonstrated statistically significant improvements with RCI 30 IU/kg in the 8-week study and RCI 60 IU/kg in the 12-week study. RCI treatment improved levels of urinary biomarkers of kidney injury (KIM-1 and OPN), expression of podoplanin, and podocyte morphology. RCI also reduced levels of desmin and fibrosis-associated collagen deposition staining. Methylprednisolone did not improve renal function or pathology in this model. CONCLUSIONS: These results provide evidence supporting the improvement of FSGS with RCI, which was superior to corticosteroid treatment in this experimental model. To the authors' knowledge, this is the first evidence that a drug for the treatment of FSGS supports podocyte recovery after repeated injury.

Absolute Configuration and Pharmacology of the Poison Frog Alkaloid Phantasmidine.

Phantasmidine, a rigid congener of the well-known nicotinic acetylcholine receptor agonist epibatidine, is found in the same species of poison frog ( Epipedobates anthonyi). Natural phantasmidine was found to be a 4:1 scalemic mixture, enriched in the (2a R,4a S,9a S) enantiomer by chiral-phase LC-MS comparison to the synthetic enantiomers whose absolute configurations were previously established by Mosher's amide analysis. The major enantiomer has the opposite S configuration at the benzylic carbon to natural epibatidine, whose benzylic carbon is R. Pharmacological characterization of the synthetic racemate and separated enantiomers established that phantasmidine is ∼10-fold less potent than epibatidine, but ∼100-fold more potent than nicotine in most receptors tested. Unlike epibatidine, phantasmidine is sharply enantioselective in its activity and the major natural enantiomer whose benzylic carbon has the 4a S configuration is more active. The stereoselective pharmacology of phantasmidine is ascribed to its rigid and asymmetric shape as compared to the nearly symmetric conformations previously suggested for epibatidine enantiomers. While phantasmidine itself is too toxic for direct therapeutic use, we believe it is a useful platform for the development of potent and selective nicotinic agonists, which may have value as pharmacological tools.

Passive accumulation of alkaloids in non-toxic frogs challenges paradigms of the origins of acquired chemical defenses.

Understanding the origins of novel, complex phenotypes is a major goal in evolutionary biology. Poison frogs of the family Dendrobatidae have evolved the novel ability to acquire alkaloids from their diet for chemical defense at least three times. However, taxon sampling for alkaloids has been biased towards colorful species, without similar attention paid to inconspicuous ones that are often assumed to be undefended. As a result, our understanding of how chemical defense evolved in this group is incomplete. Here we provide new data showing that, in contrast to previous studies, species from each undefended poison frog clade have measurable yet low amounts of alkaloids. We confirm that undefended dendrobatids regularly consume mites and ants, which are known sources of alkaloids. Further, we confirm the presence of alkaloids in two putatively non-toxic frogs from other families. Our data suggest the existence of a phenotypic intermediate between toxin consumption and sequestration-passive accumulation-that differs from active sequestration in that it involves no derived forms of transport and storage mechanisms yet results in low levels of toxin accumulation. We discuss the concept of passive accumulation and its potential role in the origin of chemical defenses in poison frogs and other toxin-sequestering organisms.

Exogenous fluorescent tracer agents based on pegylated pyrazine dyes for real-time point-of-care measurement of glomerular filtration rate.

Novel pyrazine carboxamides bearing hydrophilic poly(ethylene glycol) (PEG) moieties were designed, synthesized, and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, compounds 4d and 5c that contain about 48 ethylene oxide units in the PEG chain exhibited the most favorable physicochemical and renal clearance properties. In vitro studies show that these two compounds have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that 4d and 5c have a higher urine recovery of the injected dose than iothalamate (a commonly considered gold standard GFR agent). Pharmacokinetic studies show that these two compounds exhibit a plasma clearance equivalent to iothalamate, but with a faster (i.e. lower) terminal half-life than iothalamate (possibly from restricted distribution into the extracellular space due to large molecular size and hydrodynamic volume). Furthermore, the plasma clearance of 4d and 5c remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration exclusively. Finally, noninvasive real-time monitoring of this class of compounds was demonstrated by pharmacokinetic clearance of 5c by optical measurements in rat model, which correlates strongly with plasma concentration of the tracer. Hence, 4d and 5c are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.

Phytochemical investigation and in vitro cytotoxic evaluation of alkaloids from Abuta rufescens.

A phytochemical investigation of Abuta rufescens was performed to authenticate plant material reported previously and to assess the cytotoxicity of the alkaloids obtained from the plant. Three alkaloids which have not previously been reported from this species, two phenolic (subsessiline, an oxoaporphine, and telitoxine, an azafluoranthene) and one non-phenolic (isoimerubrine, a tropoloisoquinoline), were isolated and identified. These alkaloids, along with others previously isolated from this and another Abuta species (grandirubrine, a tropoloisoquinoline), were evaluated for cytotoxic activity against several human cancer cell lines (HCT-116 colon adenocarcinoma, ACHN renal carcinoma, and A549 lung carcinoma). The tropoloisoquinoline alkaloids (grandirubrine, imerubrine, and isoimerubrine) exhibited the greatest cytotoxicity against the cell lines, especially ACHN and HCT-116 cells. The azafluoranthene alkaloid imeluteine exhibited lesser cytotoxicity, as did one of the oxoaporphine alkaloids.

Molecular signatures of phytol-derived immunostimulants in the context of chemokine-cytokine microenvironment and enhanced immune response.

In a previous report, we observed that the phytol-derived immunostimulant, PHIS-01 (phytanol), is a nontoxic oil-in-water adjuvant which is superior to most commercial adjuvants. In contrast, the parent diterpene alcohol phytol, though highly effective as an adjuvant, is relatively toxic. To assess the importance of the polar functional group in PHIS-01, we prepared two new compounds PHIS-02 (phytanyl amine) and PHIS-03 (phytanyl mannose). All three phytol derivatives proved to be excellent adjuvants, but differed in solubility and mode of action. To delineate their molecular signatures in the local microenvironment, we performed inflammasome and cytokine microarray analyses with the peritoneal fluid of mice treated with alum or the phytol compounds above, in the presence or absence of soluble protein antigens. We report here that the phytol derivatives had a significant time-dependent impact on the host chemokine-cytokine microenvironment and subsequently on specific humoral responses. Moreover, the inclusion of protein immunogens induced further changes in host microenvironments, including rapid (<2h) expression of cytokines and chemotactic factors (IL-6, MCP-1, KC, MIP-1, and LIX), implying mobilization and activation of neutrophils, and monocytes. PHIS-01 proved to be the most effective in this regard. Inflammatory cytokine cascades were dominant even after 24h possibly to facilitate involvement of the acquired immune system with the release of B-lymphocyte chemo-attractant BLC, T-cell activation-3 chemokines TCA, IL-4, IL-12, and TIMP-1. We also noted enhanced expression of NLRP genes including NLRP3 with both alum and phytol derivatives (particularly PHIS-01).

Synthetic adjuvants for vaccine formulations: evaluation of new phytol derivatives in induction and persistence of specific immune response.

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.

Phantasmidine: an epibatidine congener from the ecuadorian poison frog Epipedobates anthonyi.

The skin of the Ecuadorian poison frog Epipedobates anthonyi contains the potent nicotinic agonists epibatidine (1) and N-methylepibatidine (3). In addition, a condensed tetracyclic epibatidine congener has been identified with activity at nicotinic acetylcholine receptors, but different selectivity than epibatidine. This rigid tetracycle has been named phantasmidine (4). Phantasmidine has a molecular formula of C(11)H(11)N(2)OCl, shares a chloropyridine moiety with 1, and also contains furan, pyrrolidine, and cyclobutane rings. A combination of GC-MS and GC-FTIR analysis with on-column derivatization, 1D NMR spectroscopy with selective irradiation, and spectral simulation, along with 2D NMR, were used to elucidate the structure from a total sample of approximately 20 microg of HPLC-purified 4 and its corresponding acetamide (5). After synthesis, this novel rigid agonist may serve as a selective probe for beta4-containing nicotinic receptors and potentially lead to useful pharmaceuticals.

Epiquinamide: a poison that wasn't from a frog that was.

In 2003, we reported the isolation, structure elucidation, and pharmacology of epiquinamide (1), a novel alkaloid isolated from an Ecuadoran poison frog, Epipedobates tricolor. Since then, several groups, including ours, have undertaken synthetic efforts to produce this compound, which appeared initially to be a novel, beta2-selective nicotinic acetylcholine receptor agonist. Based on prior chiral GC analysis of synthetic and natural samples, the absolute structure of this alkaloid was established as (1S,9aS)-1-acetamidoquinolizidine. We have synthesized the (1R*,9aS*)-isomer (epi-epiquinamide) using an iminium ion nitroaldol reaction as the key step. We have also synthesized ent-1 semisynthetically from (-)-lupinine. Synthetic epiquinamide is inactive at nicotinic receptors, in accord with recently published reports. We have determined that the activity initially reported is due to cross-contamination from co-occurring epibatidine in the isolated material.

Polyethylene glycol-based homologated ligands for nicotinic acetylcholine receptors.

A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain significant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such compounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and multivalent ligands for nAChR.

Thiophene-anthranilamides as highly potent and orally available factor Xa inhibitors.

There remains a high unmet medical need for a safe oral therapy for thrombotic disorders. The serine protease factor Xa (fXa), with its central role in the coagulation cascade, is among the more promising targets for anticoagulant therapy and has been the subject of intensive drug discovery efforts. Investigation of a hit from high-throughput screening identified a series of thiophene-substituted anthranilamides as potent nonamidine fXa inhibitors. Lead optimization by incorporation of hydrophilic groups led to the discovery of compounds with picomolar inhibitory potency and micromolar in vitro anticoagulant activity. Based on their high potency, selectivity, oral pharmacokinetics, and efficacy in a rat venous stasis model of thrombosis, compounds ZK 814048 (10b), ZK 810388 (13a), and ZK 813039 (17m) were advanced into development.

A novel P2Y(12) adenosine diphosphate receptor antagonist that inhibits platelet aggregation and thrombus formation in rat and dog models.

Irreversible platelet inhibitors, such as aspirin and clopidogrel, have limited anti-thrombotic efficacy in the clinic due to their bleeding risk. We have developed an orally active reversible P2Y(12) receptor antagonist, BX 667. The aim of this study was to determine if the reversible antagonist BX 667 had a greater therapeutic index than the irreversible P2Y(12) receptor antagonist clopidogrel. Since BX 667 is rapidly converted to its active metabolite BX 048 in rats, we first injected BX 048 intravenously (iv) in a rat arterial venous (A-V) shunt model of thrombosis. BX 048 dose- and concentration-dependently attenuated thrombosis. When administered orally, BX 667 and clopidogrel had similar efficacy, but BX 667 caused less bleeding than clopidogrel. In a rat model of a platelet-rich thrombus induced by vessel injury with FeCl(2), both BX 667 and clopidogrel exhibited higher levels of thrombus inhibition after oral administration compared to their potency in the A-V shunt model. Again, BX 667 caused less bleeding than clopidogrel. In a dog cyclic flow model, iv injection of either BX 667 or clopidogrel dose-dependently reduced thrombus formation with lower bleeding for BX 667 than clopidogrel. Inhibition of thrombosis was highly correlated with inhibition of ADP-induced platelet aggregation in these animal models. In dogs pre-treated with aspirin, BX 667 maintained its wider therapeutic index, measured by inhibition of platelet aggregation over bleeding, compared to the aspirin-clopidogrel combination. These data demonstrate that the reversible P2Y(12) receptor antagonist, BX 667, has a wider therapeutic index than clopidogrel in experimental models of thrombosis.

Characterization of beta-leptinotarsin-h and the effects of calcium flux antagonists on its activity.

beta-Leptinotarsin-h, purified from the hemolymph of the beetle Leptinotarsa haldemani, is a potent ( approximately 1 nM) neuroactive protein that rapidly (few seconds) stimulates Ca(2+) influx and neurotransmitter release. Our goals were to further characterize beta-leptinotarsin-h and to test the hypothesis that it stimulates Ca(2+) influx through presynaptic Ca(2+) channels. Analysis of partial amino acid sequences revealed that beta-leptinotarsin-h is a unique protein with significant similarity to only one other protein, the juvenile hormone esterase of Leptinotarsa decemlineata, commonly known as the Colorado potato beetle. We have examined the effect of beta-leptinotarsin-h on Ca(2+) current, Ca(2+) uptake, Ca(2+) levels, and neurotransmitter release in synaptosomes, cell lines, and neuronal systems. We found that its preferred site of action appears to be mammalian presynaptic nerve terminals. We tested antagonists of Ca(2+) flux for their effects on beta-leptinotarsin-h-stimulated Ca(2+) uptake in rat brain synaptosomes. The non-selective Ca(2+) channel blockers flunarizine, Ni(2+), ruthenium red, high-concentration thapsigargin, and SKF 96365 inhibited beta-leptinotarsin-h's activity, but none of the tested selective blockers of voltage-operated Ca(2+) channels (omega-agatoxin IVA, omega-conotoxin GVIA, omega-conotoxin MVIIC, nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand-operated, store-operated, and transduction-operated channels were also not inhibitory. beta-Leptinotarsin-h did not stimulate Na(+) uptake, ruling out Na(+) channels and many non-selective cation channels as targets. We conclude that beta-leptinotarsin-h stimulated Ca(2+) uptake through presynaptic Ca(2+) channels; which channel is yet to be determined. beta-Leptinotarsin-h may prove to be a useful tool with which to investigate calcium channels and calcium flux.

Vascular stiffness: measurements, mechanisms and implications.

Aging is the dominant process altering vascular stiffness. Risk factors for cardiovascular disease, such as smoking, hypertension and diabetes mellitus, mediate their effects by altering the structure, properties, and function of the vascular wall and endothelial components. Increased vascular stiffness exerts greater afterload stress on the heart. The ability to detect and monitor changes in the physical properties of arteries holds potential to intervene for prevention or attenuation of disease progression. Pulse wave velocity has been used as an index for vascular stiffness and as a surrogate marker for atherosclerosis in laboratory animal models and in the clinic. Mouse models have been used extensively in vascular research. We and others have developed invasive and noninvasive methods to measure pulse wave velocity in rodents, such as rats and mice. Here we review the evidence that the development of atherosclerosis contributes greatly to vascular stiffening; that endothelial nitric oxide plays an important role in modulating vascular stiffness; that angiotensin II injures the vessel and increases vascular stiffness; and that treatment with estrogen attenuates vascular inflammation and reduces vascular stiffness. In addition, we also discuss the influence of hemodynamic, metabolic, inflammatory stimuli in impairing arterial wall integrity as well as potential mechanisms modulating vascular stiffness.

Lipopolysaccharide attenuates thrombolysis in batroxobin-induced lung vasculature fibrin deposition but not in ferrous chloride-induced carotid artery thrombus in rats: role of endogenous PAI-1.

In this study, we investigated if elevation of endogenous plasminogen activator inhibitor type 1 (PAI-1) by lipopolysaccharide (LPS) can retard thrombolysis in both a rat model of lung vasculature fibrin deposition and a platelet-rich thrombus model induced by endothelial injury. By 3 h following an intravenous bolus injection of 0.5 mg/kg LPS, the plasma PAI-1 level had increased to approximately 8 ng/ml. 125I-labeled fibrinogen was injected intravenously followed by an injection of batroxobin. Batroxobin converts fibrinogen into insoluble fibrin, which was then deposited in the lungs within 5 min, followed by spontaneous fibrinolysis that completely cleared fibrin deposition in the lungs by 30 min. In rats pre-treated with LPS, spontaneous fibrinolysis was significantly retarded. In the endothelial injury model, topical application of FeCl2 on the carotid artery induced an occlusive platelet-rich thrombus, which was not sensitive to endogenous thrombolysis. Exogenous tissue-type plasminogen activator (tPA) was required to recanalize the occlusive thrombus in a dose-dependent manner. Pre-treatment with LPS did not alter the dose-response curve of exogenous tPA-induced thrombolysis. These data indicate that batroxobin-induced lung vasculature fibrin deposition in rats, unlike the FeCl2 model, is sensitive to the impact of endogenous PAI-1 on fibrinolysis.

Formal Synthesis of (+)- and (-)-Perhydrohistrionicotoxin: A "Double Henry"/Enzymatic Desymmetrization Route to the Kishi Lactam(1).

Both antipodes of the Kishi lactam (3), the versatile intermediate for the synthesis of the perhydrohistrionicotoxin (pHTX) alkaloids, have been prepared. The synthetic route involved a "double Henry" condensation between glutaraldehyde and nitroacetal 5 giving meso dioxanyldiol 4 which was acetylated and reduced to afford meso dioxane amide 8. Ultrasound-promoted deacetalization of 8 followed by Wittig olefination and reduction provided meso amide ester 10. Hydrolysis of 10 with aqueous acid followed by dehydrative cyclization with dicyclohexylcarbodiimide gave lactamdiol 11. Acetylation of 11 gave meso diacetate 2 which was an excellent substrate for esterase-mediated hydrolysis to hydroxyacetate 12. Deoxygenation of 12 using a Barton protocol, followed by Zemplén deacylation and Swern oxidation, gave the (-)-antipode of the Kishi lactam (3). Moffatt oxidation of hydroxyacetate 12 followed by ketal protection and Zemplén deacylation gave ketalalcohol 19. Barton deoxygenation of 19 followed by ketal hydrolysis gave (+)-3.

Efficacy of phytol-derived diterpenoid immunoadjuvants over alum in shaping the murine host's immune response to Staphylococcus aureus.

The ubiquitous gram-positive bacterium Staphylococcus aureus occupies a unique niche in humans for its ability to survive both as a commensal and a life-threatening pathogen. Its complex relationship with the host and its ability to engender a throng of virulence factors, have hindered the development of a successful vaccine against it. The use of immunoadjuvants to enhance host immunity and prevent the shift from commensalism to pathogenicity is a rational approach for containing infection. The objective of this study was to understand the mechanisms by which alum and two phytol-derived immunoadjuvants, phytanol (PHIS-01)(1) and phytanyl chloride (PCl)(2) shape the interaction between S. aureus and its murine host. We studied the effects of the phytol derivatives, relative to alum, on the induction of inflammatory cytokines and chemokines, recruitment of CD11b(+) cells, generation of specific anti-S. aureus antibodies and in vitro clearance of S. aureus. Our results showed that both PHIS-01 and PCl were stronger inducers of protective cytokines IL-17 and IL-1ß than alum, and far exceeded alum in enhancing anti-S. aureus antibody response. However, both alum and the phytol derivatives (particularly PCl) promoted efficient recruitment of CD11b(+) cells. Furthermore, PHIS-01, alum and to a lesser extent, PCl were able to up-regulate the expression of key inflammation-related genes that were highly down-regulated by S. aureus alone. In vitro killing assays showed that both PHIS-01 and PCl were far more potent than alum in promoting S. aureus clearance; this indicated their efficiency in shaping an effective anti-S. aureus immune microenvironment. In summary, our study provides evidence for the better effectiveness of phytol-derived immunoadjuvants over alum in enhancing anti-S. aureus immunity.