Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system

The biological activity of endocannabinoids like anandamide (AEA) and 2-arachidonoylglycerol (2-AG) is subjected in vivo to a “metabolic control”, exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K_i ∼0.6 μM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the “endocannabinoid system (ECS)”, like the AEA transporter (AMT) or CB2R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to ∼140% of controls) and inhibits the latter protein (down to ∼70%). We also show that 15-HAEA halves the synthesis of 2-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K_i ∼0.7 μM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.     

Transport of the Immunosuppressant 15-Deoxyspergualin in Human Peripheral Blood Lymphocytes

15-deoxyspergualin (DSG) is a potent immunosuppressive compound currently in clinical trials. In this study, we have characterized the uptake and intracellular localization of DSG in human peripheral blood lymphocytes (PBL′s). DSG is transported into human PBL′s and reaches an estimated maximum concentration of approximately 500μM in 6 hours. The majority of the [³H]-DSG remains in the cytoplasm of cells and that which is associated with the nucleus is only loosely associated. DSG was transported by HeLa cells, as well, suggesting uptake is not specific for hematopoietic cells. Positively charged amino acids and polyamines, which are structurally similar to DSG, were unable to compete for DSG transport suggesting that DSG is transported into cells via a pathway distinct from amino acids or polyamines.     

Synthesis and neurite growth evaluation of new analogues of honokiol, a neolignan with potent neurotrophic activity

A versatile synthetic route is reported towards the preparation of new analogues for potent neurotrophic agent biaryl-type lignan honokiol. A focused 24-membered library of derivatives containing five different groups at 5'-position of honokiol has been prepared in fair to good overall yields. Compared to the natural product, or to analogues with a short alkyl chain in this position, these new derivatives have lost most of the neurotrophic activity.     

First enantioselective syntheses of the dopamine D1 and D2 receptor modulators, (+)- and (-)-govadine

There is a pressing need to find and develop new antipsychotic agents for the treatment of schizophrenia. Current drugs primarily target dopamine D2 receptors and are only effective in the treatment of the positive symptoms of this indication. The tetrahydroprotoberberine natural product (±)-govadine has shown unique promise for the treatment of both the positive and negative symptoms of schizophrenia as it targets both dopamine D1 and D2 receptors. However, further clinical research has been hindered by the lack of availability of significant quantities of enantioenriched material. A new, enantioselective synthetic route has been developed that affords (-)-govadine in 39% overall yield over 5-steps from commercially available dopamine and homovanillic acid derivatives. Using only minor modifications in the synthetic route, (+)-govadine can be synthesized in comparable yields and enantioselectivities. The route is readily scalable as every intermediate was purified by crystallization and no flash column chromatography was necessary.     

Facile entry to (-)-(R)- and (+)-(S)-mexiletine

The title compounds, 1a and 1b, have been synthesized in a three-step sequence starting from (-)-(S) and (+)-(R)-propylene oxide, respectively, in acceptable overall yields. The enantiomeric excess values for 1a and 1b were 96% and 93% respectively, as assessed by HPLC analysis on a chiral stationary phase of the corresponding N-acetyl derivatives. The synthetic route herein presented may represent a facile entry to highly enriched mexiletine enantiomers, alternative to those previously reported in the literature.     

High affinity sialoside ligands of myelin associated glycoprotein

Myelin associated glycoprotein (Siglec-4) is a myelin adhesion receptor, that is, well established for its role as an inhibitor of axonal outgrowth in nerve injury, mediated by binding to sialic acid containing ligands on the axonal membrane. Because disruption of myelin-ligand interactions promotes axon outgrowth, we have sought to develop potent ligand based inhibitors using natural ligands as scaffolds. Although natural ligands of MAG are glycolipids terminating in the sequence NeuAcα2-3Galβ1-3(±NeuAcα2−6)GalNAcβ-R, we previously established that synthetic O-linked glycoprotein glycans with the same sequence α-linked to Thr exhibited ∼1000-fold increased affinity (∼1 μM). Attempts to increase potency by introducing a benzoylamide substituent at C-9 of the α2-3 sialic acid afforded only a two-fold increase, instead of increases of >100-fold observed for other sialoside ligands of MAG. Surprisingly, however, introduction of a 9-N-fluoro-benzoyl substituent on the α2-6 sialic acid increased affinity 80-fold, resulting in a potent inhibitor with a K_d of 15 nM. Docking this ligand to a model of MAG based on known crystal structures of other siglecs suggests that the Thr positions the glycan such that aryl substitution of the α2-3 sialic acid produces a steric clash with the GalNAc, while attaching an aryl substituent to the other sialic acid positions the substituent near a hydrophobic pocket that accounts to the increase in affinity.     

Suppression of the activities of T-lymphocyte-related enzymes in spleen by administration of an immunosuppressant, 15-deoxyspergualin

We investigated the effects of both (-) and (+)-enantiomers of 15-deoxyspergualin (DSG), an immunosuppressant, on enzyme networks in spleens of ICR mice. Of the 14 hydrolytic enzymes tested, the activity of dipeptidyl aminopeptidase IV (DAP-IV) was found to be significantly suppressed by the administration of (-)-DSG but not by that of (+)-DSG. In contrast, the activity of N-acetyl-beta-D-glucosaminidase (GlcNAc-ase) was suppressed by the administration of both enantiomers of DSG. Judging from the previous reports of ours and others which suggested specific relations of these enzymes to T-lymphocytes, the above findings may warrant further studies of these enzymes in relation to immunologic functions of the body.     

Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij and Aldrich J Peptide Res 56, 80, 2000), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar and Aldrich Org Lett 5, 613, 2003). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.     

Influence of viscous Rhodella grisea (Rhodophyceae) proteoglycan on chemically induced cough reflex

An algal extracellular biopolymer (over 8.5 × 10⁵ Da) composed of carbohydrates (52%) and protein (∼13%) has been isolated from a red alga Rhodella grisea growing in natural conditions by concentration of water medium, alcohol precipitation, dialysis and freeze-drying. This mucilagineous biopolymer contained xylose and its 3-O- and 4-O-methyl derivatives (∼63%), galactose (∼12%), glucuronic acid (11-12%), glucose (∼5%), rhamnose (∼4%), fucose (∼3-4%) and low content of others accompaning sugars. When tested on the citric acid-induced cough and reactivity of airways smooth muscle in vivo in the test system guinea pigs, this biopolymer assigned a significant cough suppressing effect. The reactivity of airways smooth muscle was not affected indicating that expectoration effect was not suppressed by biopolymer application, which is important from the pharmacological point of view.     

On the synthesis of the 2,6-dideoxysugar L-digitoxose

As deoxysugars are integral components of many natural products, the development of efficient chemical and enzymatic routes to prepare these compounds is of particular interest. Herein, we report a comparison of several synthetic methodologies used to prepare protected derivatives of the 2,6-dideoxysugar l-digitoxose. A novel, stereoselective synthetic route to efficiently access methyl 4-O-tert-butyldimethylsilyl-2,6-dideoxy-3-O-trimethylsilyl-alpha-l-ribo-hexopyranoside in 35% yield over nine facile steps is described.     

Pollination of the red-hot poker Kniphofia laxiflora (Asphodelaceae) by sunbirds

Most of the species in the large African genus Kniphofia have floral traits that conform to the bird pollination syndrome, however there has been very little empirical work to confirm that birds are effective pollinators of Kniphofia species. From selective exclusion experiments, behavioural observations and pollen load analyses, we identified Amethyst Sunbirds (Chalcomitra amethystina) and Malachite Sunbirds (Nectarinia famosa) as the primary pollinators in two populations of Kniphofia laxiflora, with bees playing a smaller role. Bird visitors obtain moderate volumes (∼ 9 µl) of relatively concentrated (∼ 15%) hexose-rich nectar in the flowers. This species is shown to be genetically self-incompatible, and thus reliant on pollinator visits for seed set. Although mountain pride butterflies (Aeropetes tulbaghia) have been identified as important pollinators of this species at two other populations, they did not occur at the two populations we studied.     

An inexpensive high-throughput nuclear magnetic resonance tube cleaning apparatus

Large-scale nuclear magnetic resonance (NMR) tube cleaning is currently a bottleneck in high-throughput NMR ligand affinity screens. Expensive alternatives include discarding the NMR tubes after a single use (∼US $2–$8/tube), using commercial NMR tube cleaners (∼$15,000), and abandoning NMR tubes for flow probe technology (∼$75,000). Instead, we describe a relatively inexpensive (∼$400) and easily constructed apparatus that can clean 180 NMR tubes per hour while using a modest amount of solvent. The application of this apparatus significantly shortens the time to recycle NMR tubes while avoiding cross-contamination and tube damage.     

Evidence for the fragmentation of VtgAb LvH in common dentex (Dentex dentex), a marine pelagophil teleost

Since the timing of the lipovitellin heavy chain (LvH) of vitellogenin Ab (VtgAb) degradation during marine teleosts ontogeny is poorly understood, the current study was planned to address some aspects of this shortcoming. Fertilized eggs (before epiboly) of Dentex dentex were analysed. Vtg-derived proteins, that appeared as 6 protein bands after SDS-PAGE, were purified using specific ion exchange chromatography. Five bands (i.e., ∼78, ∼57, ∼22, ∼19, and ∼17 kDa) were merely stained with Coomassie Blue and one band (i.e., ∼16 kDa) only with Periodic Acid Schiff (PAS). The ∼16 kDa protein band was subjected to a mass spectrometry-based sequencing. These results showed that the ∼16 kDa protein band contains a mixture of “LvH-Ab” derivatives with the same molecular weight. The specific staining system (i.e., PAS) also revealed the glycosylation of some of the LvH-Ab fragments inside the ∼16 kDa protein band. The study provides new data about fragmentation of the LvH-Ab in marine pelagophil teleosts.     

Coal fly ash and lime addition enhances the rate and efficiency of decomposition of food waste during composting

To evaluate the use of coal fly ash (CFA) on the decomposition efficiency of food waste, synthetic food waste was mixed with lime at 1.5% and 3% (equivalent to 0.94% and 1.88% CaCO₃, respectively), CFA at 5%, 10% and 15% with lime so as to achieve CaCO₃ equivalent of 1.88% and composted for 42 days in a thermophilic 20 l composter with two replicates each. Alkaline materials at 1.88% CaCO₃ equivalent successfully buffered the pH during the composting and enhanced the decomposition efficiency. When these buffering was achieved with CFA + lime, the composting period could be shortened to ∼28 days compared with ∼42 days in 3% lime. Organic decomposition in terms of CO₂ loss, carbon turnover and nitrogen transformation were significantly higher for treatments with 1.88% CaCO₃ equivalent. Nutrient transformations and compost maturity parameters indicated that addition of CFA (5–10%) with lime at 1.88% CaCO₃ equivalent enhances the decomposition efficiency and shortens the composting period by 35%.     

Synthesis of symmetric dithiophosphinic acids for “minor actinide” extraction

New aromatic dithiophosphinic acid (R₂PS₂H; DPAH) derivatives were isolated using a synthetic pathway based on nucleophilic addition at phosphorus, which leads to regiospecific aromatic substituents on phosphorus. The synthesis improves DPAH designs that can provide insight into the differences in binding/chelation between DPAH and trivalent actinides/lanthanides. The synthesis gives good yields via isolation of the DPAH as the ammonium salt. Multinuclear NMR spectroscopy and X-ray structure determination are used for the identification of both the ammonium salt and free acid of the dithiophosphinic derivatives. The DPAH products are stable when exposed to the ambient atmospheric conditions for long periods. For instance, the bis(o-trifluoromethylphenyl) DPAH derivative showed very little degradation when contacted with water and nitric acid for prolonged periods. Furthermore, this derivative selectively extracts a trivalent actinide from a lanthanide with separation factors of ∼100 000 at low pH. Overall, these DPAH derivatives are exciting new compounds for potential application in actinide and lanthanide separations technologies.     

Mechanism of Membrane Permeation Induced by Synthetic β-Hairpin Peptides

We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form β-hairpin structures under certain conditions, and a control non-β-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ∼50% of the liposomes that contain small (R_h <1.5 nm) markers, only ∼15% of those liposomes release a fluorescent dextran of 40 kDa. A multimeric model of the pore is presented based on these results. Atomistic molecular dynamics simulations show that barrels consisting of 10 β-hairpin MAX1 and MAX35 peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action.     

Modification of permeability transition pore arginine(s) by phenylglyoxal derivatives in isolated mitochondria and mammalian cells. Structure-function relationship of arginine ligands

Methylglyoxal and synthetic glyoxal derivatives react covalently with arginine residue(s) on the mitochondrial permeability transition pore (PTP). In this study, we have investigated how the binding of a panel of synthetic phenylglyoxal derivatives influences the opening and closing of the PTP. Using both isolated mitochondria and mammalian cells, we demonstrate that the resulting arginine-phenylglyoxal adduct can lead to either suppression or induction of permeability transition, depending on the net charge and hydrogen bonding capacity of the adduct. We report that phenylglyoxal derivatives that possess a net negative charge and/or are capable of forming hydrogen bonds induced permeability transition. Derivatives that were overall electroneutral and cannot form hydrogen bonds suppressed permeability transition. When mammalian cells were incubated with low concentrations of negatively charged phenylglyoxal derivatives, the addition of oligomycin caused a depolarization of the mitochondrial membrane potential. This depolarization was completely blocked by cyclosporin A, a PTP opening inhibitor, indicating that the depolarization was due to PTP opening. Collectively, these findings highlight that the target arginine(s) is functionally linked with the opening/closing mechanism of the PTP and that the electric charge and hydrogen bonding of the resulting arginine adduct influences the conformation of the PTP. These results are consistent with a model where the target arginine plays a role as a voltage sensor.     

Synthesis of bis-armed amino acid derivatives via the alkylation of ethyl isocyanoacetate and the Suzuki–Miyaura cross-coupling reaction

Summary. Two synthetic routes to bis-armed-α-amino acid derivatives are described. The first route involves alkylation of dibromo derivatives with ethyl isocyanoacetate under phase-transfer catalysis (PTC) conditions. The second route uses a palladium-mediated Suzuki–Miyaura cross-coupling reaction between a DL-4-boronophenylalanine derivative and aromatic diiodo (or dibromo) compounds.     

Effects of rapid experimental temperature increases on acute physiological stress and behaviour of stream dwelling juvenile chinook salmon

We experimentally heated small streams in summer and investigated the short-term behavioural changes and physiological stress responses of juvenile chinook salmon (Oncorhynchus tshawytscha). We rapidly raised temperatures ∼1–4 °C for 1.5 h above ambient levels of ∼7–15 °C in groundwater fed tributary streams and ∼19–23 °C in side-channel streams. Juvenile chinook rearing in groundwater fed tributaries were generally unaffected behaviourally; however, we found that temperature increase caused fish in the tributary trials to be physiologically stressed (elevations in mean cortisol concentrations ranged from 116% to 253%). Side-channel trials caused some mortality of juvenile chinook and a stronger display of behaviours indicative of stress and avoidance such as erratic swimming, abnormal posture, and aggregative behaviour. Foraging rates increased over 56 times in response to heating in side-channel trials. Cortisol levels did not increase in side-channel trials, but rather showed a trend to levels below control values suggesting an impaired stress response possibly due to chronic stress. Our results may reflect conservative responses in terms of what we may find with other salmonid species since juvenile chinook have been described as the most tolerant of the Pacific salmon species to elevated temperatures.