Concise Stereoselective Synthesis of ß-Hydroxy-γ-lactones: (4R,5R)-4-Hydroxy-γ-decalactone from the Japanese Orange Fly and Enantiomers of Arachnid Harvestmen Isolates.

The naturally occurring (4R,5R)-4-hydroxy-γ-decalactone from the Japanese orange fly and the antipode of (4S,5R)-4-hydroxy-γ-dodecalactone from the harvestmen arachnid and their stereoisomers are synthesized from the chiral pool material d-glucono-δ-lactone in a few steps. The one-pot conversion of the latter to γ-vinyl-ß-hydroxy-γ-lactone, cross-metathesis with requisite olefin, and hydrogenation enabled the synthesis of syn-lactones in just a two-pot operation. An additional efficient Pd-catalyzed allylic isomerization of γ-vinyl-ß-hydroxy-γ-lactone led to the anti-lactones in high yields.

A Novel Class of Defensive Compounds in Harvestmen: Hydroxy-γ-Lactones from the Phalangiid Egaenus convexus.

When threatened, the harvestman Egaenus convexus (Opiliones: Phalangiidae) ejects a secretion against offenders. The secretion originates from large prosomal scent glands and is mainly composed of two isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1), a ß-hydroxy-γ-lactone. The compounds were characterized by GC-MS of their microreaction derivatives, HRMS, and NMR. After the synthesis of all four possible stereoisomers of 1, followed by their separation by chiral-phase GC, the absolute configurations of the lactones in the Egaenus secretion was found to be (4S,5R)-1 (90%) and (4S,5S)-1 (10%). Hydroxy-γ-lactones represent a new class of exocrine defense compounds in harvestmen.

The mesa trail and the interacting heads motif of myosin II.

Myosin II molecules in the thick filaments of striated muscle form a structure in which the heads interact with each other and fold back onto the tail. This structure, the "interacting heads motif" (IHM), provides a mechanistic basis for the auto-inhibition of myosin in relaxed thick filaments. Similar IHM interactions occur in single myosin molecules of smooth and nonmuscle cells in the switched-off state. In addition to the interaction between the two heads, which inhibits their activity, the IHM also contains an interaction between the motor domain of one head and the initial part (subfragment 2, S2) of the tail. This is thought to be a crucial anchoring interaction that holds the IHM in place on the thick filament. S2 appears to cross the head at a specific location within a broader region of the motor domain known as the myosin mesa. Here, we show that the positive and negative charge distribution in this part of the mesa is complementary to the charge distribution on S2. We have designated this the "mesa trail" owing to its linear path across the mesa. We studied the structural sequence alignment, the location of charged residues on the surface of myosin head atomic models, and the distribution of surface charge potential along the mesa trail in different types of myosin II and in different species. The charge distribution in both the mesa trail and the adjacent S2 is relatively conserved. This suggests a common basis for IHM formation across different myosin IIs, dependent on attraction between complementary charged patches on S2 and the myosin head. Conservation from mammals to insects suggests that the mesa trail/S2 interaction plays a key role in the inhibitory function of the IHM.

Analysis of the Full-Length Pyriform Spidroin Gene Sequence.

Spiders often produce multiple types of silk, each with unique properties suiting them to certain tasks and biological functions. Orb-weaver spiders can generate more than six types of silk fibroins, with pyriform silk used to form attachment discs, adhering silk to other surfaces and substances. The unique higher-order structuring of silk fibroins has been cited as the source of their remarkable biomechanical properties. Even so, only one full-length gene sequence of pyriform silk protein 1 (PySp1) from Argiopeargentata has been reported, and studies on the mechanical properties of natural pyriform silk fibers are also lacking. To better understand the PySp1 family of genes, we used long-distance PCR (LD-PCR) to determine the sequence of PySp1 in the Araneusventricosus species. This full-length PySp1 gene is 11,931 bp in length, encoding for 3976 amino acids residues in non-repetitive N- and C-terminal domains with a central largely repetitive region made up of sixteen remarkably homogeneous units. This was similar to the previously reported A. argentata PySp1 sequence, with PySp1 from A. ventricosus also having a long repetitive N-linker that bridges the N-terminal and repetitive regions. Predictions of secondary structure and hydrophobicity of A. ventricosus PySp1 showed the pyriform silk fiber's functional properties. The amino acid compositions of PySp1 is obviously distinct from other spidroins. Our sequence makes an important contribution to understand pyriform silk protein structure and also provides a new template for recombinant pyriform silk proteins with attractive properties.

Venom collection and analysis in the pseudoscorpion Chelifer cancroides (Pseudoscorpiones: Cheliferidae).

Pseudoscorpions are very small arthropods with almost worldwide distribution. They possess a unique venom delivery system in the chelal hands of their pedipalps that has evolved independently from that of scorpions and spiders. Studies on the venom composition of pseudoscorpions are very rare. Recently, the potential venom composition of the pseudoscorpion Synsphyronus apimelus Harvey, 1987 (Pseudoscorpiones: Garypidae) has been studied by transcriptome analysis. However, a proteome analysis of venom to identify the genuine venom compounds of pseudoscorpions has not yet been performed. In our study, we have developed a non-invasive approach for extracting minute amounts of venom, which for the first time allowed collecting pure venom samples of pseudoscorpions with minimal contaminations and high reproducibility. These experiments first required a morphological investigation of the venom delivery system with a focus on the role of the lamina defensor in the release of venom. Likely, the venom delivery system of pseudoscorpions has a mechanism that prevents the release of venom if the prey is not successfully penetrated by a venom tooth. Electrical stimulation of a gland-containing chelal hand in combination with a mechanical stimulation of the lamina defensor at the base of the venom tooth resulted in an average of 5 nl of collected venom. The utility of the method was then validated by repeated venom extractions and subsequent analysis of the venom composition using MALDI-TOF mass fingerprinting. Subsequent proteomics analysis in combination with transcriptome analyses of chelal hand tissue has identified the first genuine venom compounds of pseudoscorpions with putative antimicrobial peptides. For our experiments, we used the house pseudoscorpion Chelifer cancroides (Linnaeus, 1758) (Pseudoscorpiones: Cheliferidae).

Effects of Macrothele raven venom on intrarenal invasion and metastasis of H22 liver cancer cells in mice.

BACKGROUND: Extrahepatic metastatic hepatocellular carcinoma (HCC) and its insensitivity to chemotherapy are the main causes of poor prognosis in patients with HCC. This study investigated the anti-cancer effect of Macrothele raveni venom on intrarenal metastatic HCC. MATERIALS AND METHODS: Subrenal capsule xenograft model of HCC was established by inoculation of H22 liver cancer cells. RESULTS: The general health, histology, and molecular changes were observed after administering 10 times of different dose of Macrothele raven venom injections. A volume of 0.8 µg/ml and 1.0 µg/ml of Macrothele raven venom significantly improved general health status in mice with subrenal capsule HCC tumors. Hematoxylin and eosin staining showed that Macrothele raven venom dose-dependently reduced invasion and metastasis of liver cancer cells in the kidney. Immunohistochemistry and real-time polymerase chain reaction showed that Macrothele raven venom injection dose-dependently decreased PI3K mRNA and protein, Akt protein, and mTOR mRNA expression, but increased Bad mRNA and protein expression in the kidney with H22 tumor cell invasion. 0.8 µg/ml is the most effective dose for the treatment of intrarenal metastatic HCC. CONCLUSIONS: Macrothele raven venom dose-dependently inhibits invasion and metastasis of intrarenal metastatic HCC through inhibition of PI3K-Akt-mTOR signaling and increase of Bad expression.

Mass spectrometry captures structural intermediates in protein fiber self-assembly.

Self-assembling proteins, the basis for a broad range of biological scaffolds, are challenging to study using most structural biology approaches. Here we show that mass spectrometry (MS) in combination with MD simulations captures structural features of short-lived oligomeric intermediates in spider silk formation, providing direct insights into its complex assembly process.

Longipin: An Amyloid Antimicrobial Peptide from the Harvestman Acutisoma longipes (Arachnida: Opiliones) with Preferential Affinity for Anionic Vesicles.

In contrast to vertebrate immune systems, invertebrates lack an adaptive response and rely solely on innate immunity in which antimicrobial peptides (AMPs) play an essential role. Most of them are membrane active molecules that are typically unstructured in solution and adopt secondary/tertiary structures upon binding to phospholipid bilayers. This work presents the first characterization of a constitutive AMP from the hemolymph of an Opiliones order animal: the harvestman Acutisoma longipes. This peptide was named longipin. It presents 18 aminoacid residues (SGYLPGKEYVYKYKGKVF) and a positive net charge at neutral pH. No similarity with other AMPs was observed. However, high sequence similarity with heme-lipoproteins from ticks suggested that longipin might be a protein fragment. The synthetic peptide showed enhanced antifungal activity against Candida guilliermondii and C. tropicalis yeasts (MIC: 3.8-7.5 µM) and did not interfered with VERO cells line viability at all concentrations tested (200-0.1 µM). This selectivity against microbial cells is related to the highest affinity of longipin for anionic charged vesicles (POPG:POPC) compared to zwitterionic ones (POPC), once microbial plasma membrane are generally more negatively charged compared to mammalian cells membrane. Dye leakage from carboxyfluorescein-loaded POPG:POPC vesicles suggested that longipin is a membrane active antimicrobial peptide and FT-IR spectroscopy showed that the peptide chain is mainly unstructured in solution or in the presence of POPC vesicles. However, upon binding to POPG:POPC vesicles, the FT-IR spectrum showed bands related to ß-sheet and amyloid-like fibril conformations in agreement with thioflavin-T binding assays, indicating that longipin is an amyloid antimicrobial peptide.

Predation among armored arachnids: Bothriurus bonariensis (Scorpions, Bothriuridae) versus four species of harvestmen (Harvestmen, Gonyleptidae).

Natural selection shapes prey-predator relationships and their behavioral adaptations, which seek to maximize capture success in the predator and avoidance in the prey. We tested the ability of adults of the scorpion Bothriurus bonariensis (Bothriuridae) to prey on synchronous and sympatric adults harvestmen of Acanthopachylus aculeatus, Discocyrtus prospicuus, Parampheres bimaculatus and Pachyloides thorellii (Gonyleptidae). In 72.5% of the cases B. bonariensis tried to prey on the harvestmen. The most successful captures occurred in the trials against A. aculeatus and D. prospicuus. In all the successful attacks the scorpions stung the prey between the chelicerae and consumed them, starting by the anterior portion of their bodies. The harvestmen used different defensive strategies such as fleeing before or after contact with the predator, exudating of chemical substances or staying still at the scorpion's touch. When scorpions contacted the chemical substances secreted by the harvestmen, they immediately rubbed the affected appendix against the substrate. However, exudating of chemical substances did not prevent, in any case, predation on the harvestmen. This is the first study showing the ability of scorpions to prey on different species of harvestmen, as well as the capture and defensive behaviors used by the predator and the prey.

The Scent Glands of the Neotropical Harvestman Discocyrtus pectnifemur: Morphology, Behavior and Chemistry.

Harvestmen have a pair of scent glands that open through ozopores. The literature suggests a link between the morphology of the ozopore area and the emission of a defensive secretion. A previous study on a species that aggregates in open areas, where individuals are probably more easily spotted by predators, showed that this defensive secretion causes conspecifics to flee. However, it is unknown whether this behavior occurs in species that aggregate in sheltered areas, where prey are harder to find. Herein, we describe the morphology of the ozopore area, the mode of emission of the defensive secretion, and its chemical composition in the harvestman Discocyrtus pectinifemur. We also tested if the defensive secretion is used as an alarm pheromone. We found that D. pectinifemur releases the defensive secretion in different ways, one of them being as a jet. Emission as a jet contrasts with that known for all congeners previously studied, and is in accord with the expected morphology of the ozopore. We found that the defensive secretion of D. pectinifemur does not function as an alarm pheromone. The composition of the defensive secretion, a mixture of quinones, is congruent with those already described for the clade that includes Discocyrtus. Our results support the link between the morphology of the scent glands area and the emission behavior of the defensive secretion, and they suggest that the alarm pheromone function in harvestmen may be dependent on ecological factors.

Sequential myosin phosphorylation activates tarantula thick filament via a disorder-order transition.

Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscles and a secondary (modulatory) role in striated muscles, which is regulated by Ca(2+)via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying free heads in the thick filaments that produces quick force on twitches regulated from 0 to 50% and modulation is accomplished recruiting additional force-potentiating free and blocked heads via Ca(2+)4-CaM-MLCK Ser45 phosphorylation. We have used microsecond molecular dynamics (MD) simulations of tarantula RLC NTE to understand the structural basis for phosphorylation-based regulation in tarantula thick filament activation. Trajectory analysis revealed that an inter-domain salt bridge network (R39/E58,E61) facilitates the formation of a stable helix-coil-helix (HCH) motif formed by helices P and A in the unphosphorylated NTE of both myosin heads. Phosphorylation of the blocked head on Ser45 does not induce any substantial structural changes. However, phosphorylation of the free head on Ser35 disrupts this salt bridge network and induces a partial extension of helix P along RLC helix A. While not directly participating in the HCH folding, phosphorylation of Ser35 unlocks a compact structure and allows the NTE to spontaneously undergo coil-helix transitions. The modest structural change induced by the subsequent Ser45 diphosphorylation monophosphorylated Ser35 free head facilitates full helix P extension into a single structurally stable α-helix through a network of intra-domain salt bridges (pS35/R38,R39,R42). We conclude that tarantula thick filament activation is controlled by sequential Ser35-Ser45 phosphorylation via a conserved disorder-to-order transition.

Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation.

Toxin diversity revealed by a transcriptomic study of Ornithoctonus huwena.

Spider venom comprises a mixture of compounds with diverse biological activities, which are used to capture prey and defend against predators. The peptide components bind a broad range of cellular targets with high affinity and selectivity, and appear to have remarkable structural diversity. Although spider venoms have been intensively investigated over the past few decades, venomic strategies to date have generally focused on high-abundance peptides. In addition, the lack of complete spider genomes or representative cDNA libraries has presented significant limitations for researchers interested in molecular diversity and understanding the genetic mechanisms of toxin evolution. In the present study, second-generation sequencing technologies, combined with proteomic analysis, were applied to determine the diverse peptide toxins in venom of the Chinese bird spider Ornithoctonus huwena. In total, 626 toxin precursor sequences were retrieved from transcriptomic data. All toxin precursors clustered into 16 gene superfamilies, which included six novel superfamilies and six novel cysteine patterns. A surprisingly high number of hypermutations and fragment insertions/deletions were detected, which accounted for the majority of toxin gene sequences with low-level expression. These mutations contribute to the formation of diverse cysteine patterns and highly variable isoforms. Furthermore, intraspecific venom variability, in combination with variable transcripts and peptide processing, contributes to the hypervariability of toxins in venoms, and associated rapid and adaptive evolution of toxins for prey capture and defense.

Two tarantula venom peptides as potent and differential Na(V) channels blockers.

Voltage dependent sodium (Na(V)) channels are large membrane spanning proteins which lie in the basis of action potential generation and propagation in excitable cells and hence are essential mediators of neuronal signaling. Inhibition of Na(V) channel activity is one of the core mechanisms to treat conditions related to neuronal hyperexcitability, such as epilepsy in the clinic. Na(V) channel blockers are also extensively used to locally inhibit action potential generation and related pain perceptions in the form of local anesthetics. Here we describe the isolation, biochemical characterization, synthesis and in vitro characterization of two potent Na(V) channel blockers from the venom of the Paraphysa scrofa (Phrixotrichus auratus) tarantula spider. Both Voltage sensor toxin 3 (VSTx-3, κ-theraphotoxin-Gr4a) and GTx1-15 (Toxin Gtx1-15), were originally isolated from the venom of the related tarantula Grammostola rosea and described as K(V) and Ca(V) channel blockers, respectively. In our hands, GTx1-15 was shown to be a potent inhibitor of tetrodotoxin (TTX)-sensitive channels (IC50 0.007 µM for hNa(V)1.7 and 0.12 µM for hNa(V)1.3 channels), with very little effect on TTX-resistant (Na(V)1.5 and NaV1.8) channels. VSTx-3 was demonstrated to be a potent, TTX-sensitive sodium channel blocker and especially, potent blocker of Na(V)1.8 channels (IC50 0.19 µM for hNa(V)1.3, 0.43 µM for hNa(V)1.7 and 0.77 µM for hNa(V)1.8 channels). Such potent inhibitors with differential selectivity among Na(V) channel isoforms may be used as tools to study the roles of the different channels in processes related to hyperexcitability and as lead compounds to treat pathological pain conditions.

Harvestman phenols and benzoquinones: characterisation and biosynthetic pathway.

Benzoquinones are usually present in arthropod defence exudates. Here, we describe the chemical profiles of 12 harvestman species belonging to the neotropical family Gonyleptidae. Nine of the studied species produced benzoquinones, while three produced alkyl phenols. Two benzoquinones and one phenol exhibited biological activity against bacteria and fungi. We also studied the biosynthesis of 2-ethyl-1,4-benzoquinone by feeding Magnispina neptunus individuals with ¹³C-labelled precursors; the benzoquinones were biosynthesised through a polyketide pathway using acetate and propionate building blocks.

Additional vinyl ketones and their pyranyl ketones in gonyleptid harvestmen (Arachnida: Opiliones) suggest these metabolites are widespread in this family.

Four species of gonyleptid harvestmen, Acanthogonyleptes pulcher, Gonyleptes saprophilus (Gonyleptinae), Sodreana barbiellini, and Sodreana leprevosti (Sodreaninae), were examined by GC-MS and ¹³H NMR. All of these species release vinyl ketones, and three of them produce the corresponding pyranyl ketones, which are presumed hetero-Diels-Alder (HDA) dimers. The vinyl ketones 5-methyl-1-hexen-3-one, rac-4-methyl-1-hexen-3-one, and (S)-4-methyl-1-hexen-3-one were synthesized. Natural 4-methyl-1-hexen-3-one is present as a single stereoisomer and has the R-configuration. Vinyl ketone dimers (HDA dimers) were also observed in the scent gland exudate and characterized by HRMS, ¹³C NMR, and ¹H NMR chemical shifts of the pyranyl moiety.

High conservatism in the composition of scent gland secretions in cyphophthalmid harvestmen: evidence from pettalidae.

The scent gland secretion of Austropurcellia forsteri was analyzed by gas chromatography-mass spectrometry, providing the first description of the secretion chemistry in the cyphophthalmid family Pettalidae. The secretion contained a total of 21 compounds: About 60% of the whole secretion consisted of a series of saturated, mono-unsaturated and doubly unsaturated methylketones, from C11 to C15, with a cluster of saturated and mono-unsaturated C13-methylketones dominating. A second fraction included several naphthoquinones such as 1,4-naphthoquinone (ca. 20% of secretion), 6-methyl-1,4-naphthoquinone (ca. 17%), and minor amounts of chloronaphthoquinones (ca. 2%). When compared with scent gland compositions of other representatives of cyphophthalmids (e.g. from families Sironidae and Stylocellidae), a highly conservative chemistry of cyphophthalmid secretions is apparent, based on a restricted number of methylketones and naphthoquinones.

Nitrogen-containing compounds in the scent gland secretions of European cladonychiid harvestmen (Opiliones, Laniatores, Travunioidea).

The exocrine secretions from prominently developed prosomal scent glands in four species of the European laniatorean harvestman genus Holoscotolemon (Laniatores, Travunioidea, Cladonychiidae) were analyzed by gas chromatography - mass spectrometry. Two major alkaloidal compounds were detected: Nicotine accounted for more than 97% of the secretion in Holoscotolemon jaqueti and H. oreophilum, whereas the chemically related nicotinoid alkaloid anabaseine was the major compound in H. lessiniense. In addition, a series of minor nitrogen-containing components was found, namely 3-(1-methyl-2-piperidinyl)-pyridine and anabasine, in H. jaqueti and H. oreophilum, and anabasine together with 2,3'-bipyridyl in H. lessiniense. By contrast, extracts of adult H. unicolor did not show any components. In one juvenile specimen of H. unicolor, however, low amounts of alkyl pyrazines (dimethyl-isobutyl- and dimethyl-isopentylpyrazines) were detected. Nitrogen-containing components previously were found in Sclerobunus robustus (an American travunioid harvestman), so scent gland-derived alkaloids may be widespread or even common in the Travunioidea. Alkaloids have not been reported for other opilionid scent gland secretions outside the Travunioidea, and we hypothesize that they may be the phylogenetically ancestral allomones in the Laniatores, having been reduced and replaced by a phenol- and benzoquinone-rich chemistry in the more derived grassatorean taxa.

6-Alkyl-3,4-dihydro-2H-pyrans: chemical secretion compounds in neotropical harvestmen.

The defensive secretions of five neotropical species of harvestmen (Opiliones: Gonyleptidae) from the Brazilian Atlantic Forest were analyzed and chemically characterized by GC-MS and NMR methods. Three of the species, Cobania picea, Roweria virescens, and Serracutisoma proximum, secrete a mixture of 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone. The secretions produced by the other two species, Iporangaia pustulosa and Neosadocus maximus, contain 1-hepten-3-one, 5-methyl-1-hexen-3-one, and 1-(6-butyl-3,4-dihydro-2H-pyran-2-yl)pentanone (1) as major components, as well as 2,3-dimethyl-1,4-benzoquinone and 2-ethyl-3-methyl-1,4-benzoquinone as minor constituents. The dihydropyran 1-(6-butyl-3,4-dihydro-2H-pyran-2-yl)pentanone (1) is a new natural product, composed of two 1-hepten-3-one subunits formally linked in a hetero-Diels-Alder reaction. The natural product was proven to be racemic, and its biogenetic origin is discussed.

Naphthoquinones and anthraquinones from scent glands of a dyspnoid Harvestman, Paranemastoma quadripunctatum.

Extracts of Paranemastoma quadripunctatum (Opiliones, Dyspnoi, Nemastomatidae) contained seven components, all of which likely originated from the secretion of well-developed prosomal scent glands. The two main components (together accounting for more than 90% of the secretion) were identified as 1,4-naphthoquinone and 6-methyl-1,4-naphthoquinone. The minor components were 1,4-naphthalenediol, two methoxy-naphthoquinones (2-methoxy-1,4-naphthoquinone, and 2-methoxy-6-methyl-1,4-naphthoquinone) and two anthraquinones (2-methyl-9,10-anthraquinone and a dimethyl-9,10-anthraquinone). While some chemical data on scent gland secretions of the other suborders of Opiliones (Cyphophthalmi, palpatorean Eupnoi, and Laniatores) already exist, this is the first report on the scent gland chemistry in the Dyspnoi. Naphthoquinones are known scent gland exudates of Cyphophthalmi and certain Eupnoi, methoxy-naphthoquinones and anthraquinones are new for opilionid scent gland secretions.