New Pyridyl and Dihydroisoquinoline Alkaloids Isolated from the Chevron Nemertean Amphiporus angulatus.

Nemertean worms contain toxins that are used to paralyze their prey and to deter potential predators. Hoplonemerteans often contain pyridyl alkaloids like anabaseine that act through nicotinic acetylcholine receptors and crustacean chemoreceptors. The chemical reactivity of anabaseine, the first nemertean alkaloid to be identified, has been exploited to make drug candidates selective for alpha7 subtype nAChRs. GTS-21, a drug candidate based on the anabaseine scaffold, has pro-cognitive and anti-inflammatory actions in animal models. The circumpolar chevron hoplonemertean Amphiporus angulatus contains a multitude of pyridyl compounds with neurotoxic, anti-feeding, and anti-fouling activities. Here, we report the isolation and structural identification of five new compounds, doubling the number of pyridyl alkaloids known to occur in this species. One compound is an isomer of the tobacco alkaloid anatabine, another is a unique dihydroisoquinoline, and three are analogs of the tetrapyridyl nemertelline. The structural characteristics of these ten compounds suggest several possible pathways for their biosynthesis.

Highly N-Methylated Peptides from the Antarctic Sponge Inflatella coelosphaeroides Are Active against Plasmodium falciparum.

Malaria, caused by the parasite Plasmodium falciparum, continues to threaten much of the world's population, and there is a pressing need for expanding treatment options. Natural products have been a vital source of such drugs, and here we report seven new highly N-methylated linear peptides, friomaramide B (2) and shagamides A-F (3-8) from the marine sponge Inflatella coelosphaeroides, collected in Antarctic waters, which demonstrate activity against three strains of blood-stage P. falciparum. The planar structures of these metabolites were solved by interpreting NMR data, as well as HRESIMS/MS fragmentation patterns, while Marfey's analysis was used to establish the configurations of the amino acids. Reisolation of the previously reported compound friomaramide A (1) allowed us to revise its structure. The panel of isolated compounds allowed establishing structure/activity relationships and provided information for future structure optimization for this class of P. falciparum inhibitory metabolites.

A Methyl Scan of the Pyrrolidinium Ring of Nicotine Reveals Significant Differences in Its Interactions with α7 and α4ß2 Nicotinic Acetylcholine Receptors.

The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the α4ß2 and α7 subtypes. A "methyl scan" of the pyrrolidinium ring was used to detect differences in nicotine's interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in nicotine's receptor interactions. Replacing the 1'-N-methyl with an ethyl group or adding a second 1'-N-methyl group significantly reduced interaction with α4ß2 but not α7 receptors. The 2'-methylation uniquely enhanced binding and agonist potency at α7 receptors. Although 3'- and 5'-trans-methylations were much better tolerated by α7 receptors than α4ß2 receptors, 4'-methylation decreased potency and efficacy at α7 receptors much more than at α4ß2 receptors. Whereas cis-5'-methylnicotine lacked agonist activity and displayed a low affinity at both receptors, trans-5'-methylnicotine retained considerable α7 receptor activity. Differences between the two 5'-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine analog A84543 were consistent with what was found for the 5'-methylnicotines. Computer docking of the methylnicotines to the Lymnaea acetylcholine binding protein crystal structure containing two persistent waters predicted most of the changes in receptor affinity that were observed with methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller effects of 1'-, 3'-, and 5'-methylations and the greater effects of 2'- and 4'-methylations on nicotine α7 nAChR interaction might be exploited for the design of new drugs based on the nicotine scaffold. SIGNIFICANCE STATEMENT: Using a comprehensive "methyl scan" approach, we show that the orthosteric binding sites for acetylcholine and nicotine in the two major brain nicotinic acetylcholine receptors interact differently with the pyrrolidinium ring of nicotine, and we suggest reasons for the higher affinity of nicotine for the heteromeric receptor. Potential sites for nicotine structure modification were identified that may be useful in the design of new drugs targeting these receptors.

Discovery of new pyrimidine-5-carbonitrile derivatives as anticancer agents targeting EGFRWT and EGFRT790M.

A new series of pyrimidine-5-carbonitrile derivatives has been designed as ATP mimicking tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR). These compounds were synthesized and evaluated for their in vitro cytotoxic activities against a panel of four human tumor cell lines, namely colorectal carcinoma (HCT-116), hepatocellular carcinoma (HepG-2), breast cancer (MCF-7), and non-small cell lung cancer cells (A549). Five of the synthesized compounds, 11a, 11b, 12b, 15b and 16a, were found to exhibit moderate antiproliferative activity against the tested cell lines and were more active than the EGFR inhibitor erlotinib. In particular, compound 11b showed 4.5- to 8.4-fold erlotinib activity against HCT-116, HepG-2, MCF-7, and A549 cells with IC50 values of 3.37, 3.04, 4.14, and 2.4 µM respectively. Moreover, the most cytotoxic compounds that showed promising IC50 values against the four cancer cell lines were subjected to further investigation for their kinase inhibitory activities against EGFRWT and EGFRT790M using homogeneous time resolved fluorescence (HTRF) assay. Compound 11b was also found to be the most active compound against both EGFRWT and mutant EGFRT790M, exhibiting IC50 values of 0.09 and 4.03 µM, respectively. The cell cycle and apoptosis analyses revealed that compound 11b can arrest the cell cycle at the G2/M phase and induce significant apoptotic effects in HCT-116, HepG-2, and MCF-7 cells. Additionally, compound 11b upregulated the level of caspase-3 by 6.5 fold in HepG-2 when compared with the control. Finally, molecular docking studies were carried out to examine the binding mode of the synthesized compounds against the proposed targets; EGFRWT and EGFRT790M. Additional in silico ADMET studies were performed to explore drug-likeness properties.

Synthesis and Characterization of Tungsten Nitrido Amido Guanidinato Complexes as Precursors for Chemical Vapor Deposition of WNxCy Thin Films.

Tungsten nitrido amido guanidinato complexes of the type WN(NR2)[(NR')2C(NR2)]2 (R = Me, Et; R' = i Pr, Cy) were synthesized as precursors for aerosol-assisted chemical vapor deposition (AACVD) of WNxCy thin films. The reaction of tungsten nitrido amido complexes of the type WN(NR2)3 (R = Me, Et) with two equivalents of a carbodiimide R'N=C=NR' (R' = i Pr, Cy) resulted in two insertions of a carbodiimide into W-N(amido) bonds, affording bis(guanidinato) amido nitrido tungsten complexes. These compounds were characterized by 14N NMR, indicating distinctive chemical shifts for each type of N-bound ligand. Crystallographic structure determination of WN(NMe2)[(N i Pr)2C(NMe2)]2 showed the guanidinato ligands to be non-equivalent. The complex WN(NMe2)[(N i Pr)2C(NMe2)]2 was demonstrated to serve as a precursor for AACVD of WNxCy thin films, resulting in featureless, X-ray amorphous thin films for growth temperatures 200 - 400 °C.

Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity.

The hepatic tricarboxylic acid (TCA) cycle is central to integrating macronutrient metabolism and is closely coupled to cellular respiration, free radical generation, and inflammation. Oxidative flux through the TCA cycle is induced during hepatic insulin resistance, in mice and humans with simple steatosis, reflecting early compensatory remodeling of mitochondrial energetics. We hypothesized that progressive severity of hepatic insulin resistance and the onset of nonalcoholic steatohepatitis (NASH) would impair oxidative flux through the hepatic TCA cycle. Mice (C57/BL6) were fed a high-trans-fat high-fructose diet (TFD) for 8 wk to induce simple steatosis and NASH by 24 wk. In vivo fasting hepatic mitochondrial fluxes were determined by(13)C-nuclear magnetic resonance (NMR)-based isotopomer analysis. Hepatic metabolic intermediates were quantified using mass spectrometry-based targeted metabolomics. Hepatic triglyceride accumulation and insulin resistance preceded alterations in mitochondrial metabolism, since TCA cycle fluxes remained normal during simple steatosis. However, mice with NASH had a twofold induction (P< 0.05) of mitochondrial fluxes (µmol/min) through the TCA cycle (2.6 ± 0.5 vs. 5.4 ± 0.6), anaplerosis (9.1 ± 1.2 vs. 16.9 ± 2.2), and pyruvate cycling (4.9 ± 1.0 vs. 11.1 ± 1.9) compared with their age-matched controls. Induction of the TCA cycle activity during NASH was concurrent with blunted ketogenesis and accumulation of hepatic diacylglycerols (DAGs), ceramides (Cer), and long-chain acylcarnitines, suggesting inefficient oxidation and disposal of excess free fatty acids (FFA). Sustained induction of mitochondrial TCA cycle failed to prevent accretion of "lipotoxic" metabolites in the liver and could hasten inflammation and the metabolic transition to NASH.

A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis.

Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multi-step pathway. The early intermediates of this pathway are notoriously reactive and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. In the present paper, we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA respectively). We present cheminformatic, biochemical, genetic and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis, yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multi-enzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites.

The role of select subtype polymorphisms on HIV-1 protease conformational sampling and dynamics.

HIV-1 protease is an essential enzyme for viral particle maturation and is a target in the fight against HIV-1 infection worldwide. Several natural polymorphisms are also associated with drug resistance. Here, we utilized both pulsed electron double resonance, also called double electron-electron resonance, and NMR (15)N relaxation measurements to characterize equilibrium conformational sampling and backbone dynamics of an HIV-1 protease construct containing four specific natural polymorphisms commonly found in subtypes A, F, and CRF_01 A/E. Results show enhanced backbone dynamics, particularly in the flap region, and the persistence of a novel conformational ensemble that we hypothesize is an alternative flap orientation of a curled open state or an asymmetric configuration when interacting with inhibitors.

Phenazine antibiotic inspired discovery of potent bromophenazine antibacterial agents against Staphylococcus aureus and Staphylococcus epidermidis.

Nearly all clinically used antibiotics have been (1) discovered from microorganisms (2) using phenotype screens to identify inhibitors of bacterial growth. The effectiveness of these antibiotics is attributed to their endogenous roles as bacterial warfare agents against competing microorganisms. Unfortunately, every class of clinically used antibiotic has been met with drug resistant bacteria. In fact, the emergence of resistant bacterial infections coupled to the dismal pipeline of new antibacterial agents has resulted in a global health care crisis. There is an urgent need for innovative antibacterial strategies and treatment options to effectively combat drug resistant bacterial pathogens. Here, we describe the implementation of a Pseudomonas competition strategy, using redox-active phenazines, to identify novel antibacterial leads against Staphylococcus aureus and Staphylococcus epidermidis. In this report, we describe the chemical synthesis and evaluation of a diverse 27-membered phenazine library. Using this microbial warfare inspired approach, we have identified several bromophenazines with potent antibacterial activities against S. aureus and S. epidermidis. The most potent bromophenazine analogue from this focused library demonstrated a minimum inhibitory concentration (MIC) of 0.78-1.56 µM, or 0.31-0.62 µg mL(-1), against S. aureus and S. epidermidis and proved to be 32- to 64-fold more potent than the phenazine antibiotic pyocyanin in head-to-head MIC experiments. In addition to the discovery of potent antibacterial agents against S. aureus and S. epidermidis, we also report a detailed structure-activity relationship for this class of bromophenazine small molecules.

Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize.

Phytoalexins constitute a broad category of pathogen- and insect-inducible biochemicals that locally protect plant tissues. Because of their agronomic significance, maize and rice have been extensively investigated for their terpenoid-based defenses, which include insect-inducible monoterpene and sesquiterpene volatiles. Rice also produces a complex array of pathogen-inducible diterpenoid phytoalexins. Despite the demonstration of fungal-induced ent-kaur-15-ene production in maize over 30 y ago, the identity of functionally analogous maize diterpenoid phytoalexins has remained elusive. In response to stem attack by the European corn borer (Ostrinia nubilalis) and fungi, we observed the induced accumulation of six ent-kaurane-related diterpenoids, collectively termed kauralexins. Isolation and identification of the predominant Rhizopus microsporus-induced metabolites revealed ent-kaur-19-al-17-oic acid and the unique analog ent-kaur-15-en-19-al-17-oic acid, assigned as kauralexins A3 and B3, respectively. Encoding an ent-copalyl diphosphate synthase, fungal-induced An2 transcript accumulation precedes highly localized kauralexin production, which can eventually exceed 100 µg · g(-1) fresh weight. Pharmacological applications of jasmonic acid and ethylene also synergize the induced accumulation of kauralexins. Occurring at elevated levels in the scutella of all inbred lines examined, kauralexins appear ubiquitous in maize. At concentrations as low as 10 µg · mL(-1), kauralexin B3 significantly inhibited the growth of the opportunistic necrotroph R. microsporus and the causal agent of anthracnose stalk rot, Colletotrichum graminicola. Kauralexins also exhibited significant O. nubilalis antifeedant activity. Our work establishes the presence of diterpenoid defenses in maize and enables a more detailed analysis of their biosynthetic pathways, regulation, and crop defense function.

A folding motif formed with an expanded genetic alphabet.

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ-:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ-:dZ pairs join parallel strands in a four-stranded compact down-up-down-up fold. These have two possible structures: one with intercalated dZ-:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC+-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis.

A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

Discovery of the Nicotinic Receptor Toxin Anabaseine in a Polystiliferan Nemertean.

Nemerteans (also called Nemertines) are a phylum of predominantly marine worms that use toxins to capture prey and to defend themselves against predators. Hoplonemerteans have a proboscis armed with one or more stylets used in prey capture and are taxonomically divided into Order Monostilifera, whose members possess a single large proboscis stylet, and Order Polystilifera, whose members have multiple small stylets. Many monostiliferans contain alkaloidal toxins, including anabaseine, that stimulate and then desensitize nicotinic acetylcholine receptors that are present in all animals. These compounds also interact with pyridyl chemoreceptors in crustaceans, reducing predation and larval settlement. Anabaseine has been a lead compound in the design of alpha7 nicotinic acetylcholine receptor agonists like GTS-21 (also called DMXBA) to treat disorders of cognition such as Alzheimer's disease and schizophrenia. These drug candidates also display anti-inflammatory activities of potential medical importance. Most polystiliferans live deep in open oceans and are relatively inaccessible. We fortunately obtained two live specimens of a large benthic polystiliferan, Paradrepanophorus crassus (Pc), from the coast of Spain. MS and NMR analyses of the Ehrlich's reagent derivative allowed identification of anabaseine. A spectrophotometric assay for anabaseine, also based on its reaction with Ehrlich's reagent, revealed high concentrations of anabaseine in the body and proboscis. Apparently, the biosynthetic mechanism for producing anabaseine was acquired early in the evolution of the Hoplonemertea, before the monostiliferan-polystiliferan divergence.

Evidence that the folate-dependent proteins YgfZ and MnmEG have opposing effects on growth and on activity of the iron-sulfur enzyme MiaB.

The folate-dependent protein YgfZ of Escherichia coli participates in the synthesis and repair of iron-sulfur (Fe-S) clusters; it belongs to a family of enzymes that use folate to capture formaldehyde units. Ablation of ygfZ is known to reduce growth, to increase sensitivity to oxidative stress, and to lower the activities of MiaB and other Fe-S enzymes. It has been reported that the growth phenotype can be suppressed by disrupting the tRNA modification gene mnmE. We first confirmed the latter observation using deletions in a simpler, more defined genetic background. We then showed that deleting mnmE substantially restores MiaB activity in ygfZ deletant cells and that overexpressing MnmE with its partner MnmG exacerbates the growth and MiaB activity phenotypes of the ygfZ deletant. MnmE, with MnmG, normally mediates a folate-dependent transfer of a formaldehyde unit to tRNA, and the MnmEG-mediated effects on the phenotypes of the ΔygfZ mutant apparently require folate, as evidenced by the effect of eliminating all folates by deleting folE. The expression of YgfZ was unaffected by deleting mnmE or overexpressing MnmEG or by folate status. Since formaldehyde transfer is a potential link between MnmEG and YgfZ, we inactivated formaldehyde detoxification by deleting frmA. This deletion had little effect on growth or MiaB activity in the ΔygfZ strain in the presence of formaldehyde, making it unlikely that formaldehyde alone connects the actions of MnmEG and YgfZ. A more plausible explanation is that MnmEG erroneously transfers a folate-bound formaldehyde unit to MiaB and that YgfZ reverses this.

Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize.

Nonvolatile terpenoid phytoalexins occur throughout the plant kingdom, but until recently were not known constituents of chemical defense in maize (Zea mays). We describe a novel family of ubiquitous maize sesquiterpenoid phytoalexins, termed zealexins, which were discovered through characterization of Fusarium graminearum-induced responses. Zealexins accumulate to levels greater than 800 µg g⁻¹ fresh weight in F. graminearum-infected tissue. Their production is also elicited by a wide variety of fungi, Ostrinia nubilalis herbivory, and the synergistic action of jasmonic acid and ethylene. Zealexins exhibit antifungal activity against numerous phytopathogenic fungi at physiologically relevant concentrations. Structural elucidation of four members of this complex family revealed that all are acidic sesquiterpenoids containing a hydrocarbon skeleton that resembles ß-macrocarpene. Induced zealexin accumulation is preceded by increased expression of the genes encoding TERPENE SYNTHASE6 (TPS6) and TPS11, which catalyze ß-macrocarpene production. Furthermore, zealexin accumulation displays direct positive relationships with the transcript levels of both genes. Microarray analysis of F. graminearum-infected tissue revealed that Tps6/Tps11 were among the most highly up-regulated genes, as was An2, an ent-copalyl diphosphate synthase associated with production of kauralexins. Transcript profiling suggests that zealexins cooccur with a number of antimicrobial proteins, including chitinases and pathogenesis-related proteins. In addition to zealexins, kauralexins and the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-glucose) were produced in fungal-infected tissue. HDMBOA-glucose accumulation occurred in both wild-type and benzoxazine-deficient1 (bx1) mutant lines, indicating that Bx1 gene activity is not required for HDMBOA biosynthesis. Together these results indicate an important cooperative role of terpenoid phytoalexins in maize biochemical defense.

Ring Distortion of Vincamine Leads to the Identification of Re-Engineered Antiplasmodial Agents.

There is a significant need for new agents to combat malaria, which resulted in ∼409,000 deaths globally in 2019. We utilized a ring distortion strategy to create complex and diverse compounds from vincamine with the goal of discovering molecules with re-engineered biological activities. We found compound 8 (V3b) to target chloroquine-resistant Plasmodium falciparum Dd2 parasites (EC50 = 1.81 ± 0.09 µM against Dd2 parasites; EC50 > 40 µM against HepG2 cells) and established structure-activity relationships for 25 related analogues. New analogue 30 (V3ss, Dd2, EC50 = 0.25 ± 0.004 µM; HepG2, EC50 > 25 µM) was found to demonstrate the most potent activity, which prevents exit on the parasite from the schizont stage of intraerythrocytic development and requires >24 h to kill P. falciparum Dd2 cells. These findings demonstrate the potential that vincamine ring distortion has toward the discovery of novel antimalarial agents and other therapies significant to human health.

Preventing Morphine-Seeking Behavior through the Re-Engineering of Vincamine's Biological Activity.

Innovative discovery strategies are essential to address the ongoing opioid epidemic in the United States. Misuse of prescription and illegal opioids (e.g., morphine, heroin) has led to major problems with addiction and overdose. We used vincamine, an indole alkaloid, as a synthetic starting point for dramatic structural alterations of its complex, fused ring system to synthesize 80 diverse compounds with intricate molecular architectures. A select series of vincamine-derived compounds were screened for both agonistic and antagonistic activities against a panel of 168 G protein-coupled receptor (GPCR) drug targets. Although vincamine was without an effect, the novel compound 4 (V2a) demonstrated antagonistic activities against hypocretin (orexin) receptor 2. When advanced to animal studies, 4 (V2a) significantly prevented acute morphine-conditioned place preference (CPP) and stress-induced reinstatement of extinguished morphine-CPP in mouse models of opioid reward and relapse. These results demonstrate that the ring distortion of vincamine offers a promising way to explore new chemical space of relevance to opioid addiction.

Effects of soldier-derived terpenes on soldier caste differentiation in the termite Reticulitermes flavipes.

Primer pheromones play key roles in regulating division of labor, which is a fundamental and defining aspect of insect sociality. Primer pheromones are chemical messengers that transmit hormone-like messages among colony members; in recipients, these messages can either induce or suppress phenotypic caste differentiation. Here, we investigated soldier caste-derived chemicals as possible primer pheromones in the lower termite Reticulitermes flavipes, a species for which no primer pheromones have yet been identified. We determined that soldier head extracts (SHE), when provided to totipotent workers along with the insect morphogenetic juvenile hormone (JH), significantly enhanced soldier caste differentiation. When applied alone, however, SHE had no impacts on caste differentiation, survivorship, or any other aspect of worker biology. These findings support a function of soldier chemicals as primer pheromones that enhance the action of the endogenous JH. In accord with previous studies, gamma-cadinene and the corresponding aldehyde, gamma-cadinenal, were identified by gas chromatography-mass spectrometry and nuclear magnetic resonance analyses as the two most abundant components of R. flavipes SHE. Validative bioassays with commercially available cadinene confirmed activity. Several other terpenes, previously identified in R. flavipes soldiers, also were found to be active. These findings reveal a novel primer pheromone-like function for soldier-derived terpenes in termites and further suggest convergent evolution of terpene functions in enhancing JH-dependent soldier caste differentiation.

Total structure determination of grassypeptolide, a new marine cyanobacterial cytotoxin.

A collection of the cyanobacterium Lyngbya confervoides off Grassy Key in Florida yielded grassypeptolide (1), a 31-membered macrocyclic depsipeptide with unusually high D-amino acid content, two thiazolines, and one beta-amino acid. We report the rigorous 3D structure determination and conformational analysis in solution and solid state by NMR, MS, X-ray crystallography, chemical degradation, and molecular modeling involving distance geometry and restrained molecular dynamics. Grassypeptolide (1) inhibited cancer cell growth with IC50 values from 1.0 to 4.2 microM.

Cold adaptation does not alter ATP homeostasis during cold exposure in Drosophila melanogaster.

In insects and other ectotherms, cold temperatures cause a coma resulting from loss of neuromuscular function, during which ionic and metabolic homeostasis are progressively lost. Cold adaptation improves homeostasis during cold exposure, but the ultimate targets of selection are still an open question. Cold acclimation and adaptation remodels mitochondrial metabolism in insects, suggesting that aerobic energy production during cold exposure could be a target of selection. Here, we test the hypothesis that cold adaptation improves the ability to maintain rates of aerobic energy production during cold exposure by using 31 P NMR on live flies. Using lines of Drosophila melanogaster artificially selected for fast and slow recovery from a cold coma, we show that cold exposure does not lower ATP levels and that cold adaptation does not alter aerobic ATP production during cold exposure. Cold-hardy and cold-susceptible lines both experienced a brief transition to anaerobic metabolism during cooling, but this was rapidly reversed during cold exposure, suggesting that oxidative phosphorylation was sufficient to meet energy demands below the critical thermal minimum, even in cold-susceptible flies. We thus reject the hypothesis that performance under mild low temperatures is set by aerobic ATP supply limitations in D. melanogaster, excluding oxygen and capacity limitation as a weak link in energy supply. This work suggests that the modulations to mitochondrial metabolism resulting from cold acclimation or adaptation may arise from selection on a biosynthetic product(s) of those pathways rather than selection on ATP supply during cold exposure.