Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis. SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 μM and is found at concentrations between 1 and 10 μM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 μM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 μM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 μM. We interpret these results to suggest that B. dendrobatidis produces an “armamentarium” of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.
Twenty-one mono- and biscationic quaternary ammonium amphiphiles (monoQACs and bisQACs) were rapidly prepared in order to investigate the effects of rigidity of a diamine core structure on antiseptic activity. As anticipated, bioactivity against a panel of 6 bacteria including MRSA strains was strong for bisQAC structures, and clearly correlated to the length of non-polar side chains. Modest advantages were noted for amide-containing side chains, as compared to straight-chained alkyl substituents. Surprisingly, antiseptics with more rigidly disposed side chains, such as those in DABCO-12,12, showed the highest level of antimicrobial activity, with single-digit MIC values or better against the entire bacterial panel, including submicromolar activity against a MRSA strain.
Premise of the StudyUnderstanding the phylogenetic distribution of defensive plant secondary metabolites is essential to the macroevolutionary study of chemically mediated plant–animal interactions. The chemical ecology of pyrrolizidine alkaloids (PAs) has been extensively studied in a number of plant–herbivore systems, including Apocynaceae (the milkweed and dogbane family) and Danainae (the milkweed and clearwing butterflies). A systematic survey is necessary to establish a detailed understanding of their occurrence across Apocynaceae. A survey of this species‐rich, mainly tropical and subtropical family will rely heavily on small tissue samples removed from herbarium specimens, some of which will be very old and/or preserved with alcohols or mercuric chloride.MethodsWe optimized PA extraction methods from small leaf fragments of recently collected silica‐dried leaves of the PA‐positive Echites umbellatus, varying crushing and extraction time. We then applied our optimized method to leaf fragments from 70–167‐year‐old herbarium specimens of E. umbellatus. To simulate the effect of alcohol treatment on PA detectability in herbarium specimens, we incubated freshly collected leaves of the PA‐positive Parsonsia alboflavescens in three different alcohols before drying and compared PA recovery to freshly dried controls. PAs were quantified using high‐performance liquid chromatography–mass spectrometry analysis. X‐ray fluorescence was used to identify mercury‐containing specimens.ResultsFifteen seconds of leaf crushing followed by 24 h of extraction were optimal for PA free‐base and N‐oxide recovery. This method yielded ~50‐fold greater PA recovery than prior methods. Half of the herbarium specimens (13 of 23), including the oldest, tested positive for PAs; leaf age did not correlate with success in PA extraction. Treatment of fresh leaves with alcohol before drying did not diminish PA recovery; mercury was observed in both PA‐positive and PA‐negative specimens.Conclusions PAs can be reliably detected in small tissue samples from herbarium specimens up to 167 years old, including specimens that had been treated with alcohol or mercury salts. The variability of PA presence among herbarium specimens of E. umbellatus indicates that multiple specimens will need to be tested before a particular species is determined to lack PAs.
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