��-Conotoxin GVIA Mimetics that Bind and Inhibit Neuronal Cav2.2 Ion Channels

The neuronal voltage-gated N-type calcium channel (Ca_v2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived ω-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a ¹²⁵I-ω-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca_v2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of ω-conotoxin GVIA and found to be active in the ¹²⁵I-ω-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.     

Design and Synthesis of Novel Xyloketal Derivatives and Their Protective Activities against H2O2-Induced HUVEC Injury

In this work, we designed and synthesized a series of amide derivatives (1–13), benzoxazine derivatives (16–28) and amino derivatives (29–30) from xyloketal B. All 28 new derivatives and seven known compounds (14, 15, 31–35) were evaluated for their protection against H₂O₂-induced HUVEC injury. 23 and 24 exhibited more potential protective activities than other derivatives; and the EC₅₀ values of them and the leading compound 31 (xyloketal B) were 5.10, 3.59 and 15.97 μM, respectively. Meanwhile, a comparative molecular similarity indices analysis (CoMSIA) was constructed to explain the structural activity relationship of these xyloketal derivatives. This 3D QSAR model from CoMSIA suggested that the derived model exhibited good predictive ability in the external test-set validation. Derivative 24 fit well with the COMSIA map, therefore it possessed the highest activity of all compounds. Compounds 23, 24 and 31 (xyloketal B) were further to examine in the JC-1 mitochondrial membrane potential (MMP) assay of HUVECs using flow cytometry (FCM). The result indicated that 23 and 24 significantly inhibited H₂O₂-induced decrease of the cell mitochondrial membrane potential (ΔΨm) at 25 μM. Collectively, the protective effects of xyloketals on H₂O₂-induced endothelial cells may be generated from oxidation action by restraining ROS and reducing the MMP.     

Inhibition of N-Type Calcium Channels by Fluorophenoxyanilide Derivatives

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Ca_v2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.     

Selective Blocking Effects of 4,9-Anhydrotetrodotoxin,Purified from a Crude Mixture of Tetrodotoxin Analogues,on NaV1.6 Channels and Its Chemical Aspects

Tetrodotoxin (TTX) is a potent neurotoxin found in a number of marine creatures including the pufferfish, where it is synthesized by bacteria and accumulated through the food chain. It is a potent and selective blocker of some types of voltage-gated Na⁺ channel (Na_V channel). 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX) was purified from a crude mixture of TTX analogues (such as TTX, 4-epiTTX, 6-epiTTX, 11-oxoTTX and 11-deoxyTTX) by the use of liquid chromatography-fluorescence detection (LC-FLD) techniques. Recently, it has been reported that 4,9-anhydroTTX selectively blocks the activity of Na_V1.6 channels with a blocking efficacy 40–160 times higher than that for other TTX-sensitive Na_V1.x channel isoforms. However, little attention has been paid to the molecular properties of the α-subunit in Na_V1.6 channels and the characteristics of binding of 4,9-anhydroTTX. From a functional point of view, it is important to determine the relative expression of Na_V1.6 channels in a wide variety of tissues. The aim of this review is to discuss briefly current knowledge about the pharmacology of 4,9-anhydroTTX, and provide an analysis of the molecular structure of native Na_V1.6 channels. In addition, chemical aspects of 4,9-anhydroTTX are briefly covered.     

Marine Toxins Targeting Ion Channels

This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs), as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs), are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na⁺ (Na_V), Ca²⁺ (Ca_V), and K⁺ (K_V) channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR), and the ATP-activated (P2X_nR) receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of Na_V, Ca_V, or K_V channels opens a pore that is specific for Na⁺, Ca²⁺, or K⁺, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2X_nRs allows the passage of cations (e.g., Na⁺, K⁺, and/or Ca²⁺), whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl⁻ and/or HCO₃⁻). In this regard, the activation of Na_V and Ca_V as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of K_V as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers) of ion channel functions to treat or to alleviate a specific ion channel-linked disease (e.g., channelopaties).     

Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1)

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na⁺]_I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca²⁺) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca²⁺-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.     

Aspergillus Sydowii Marine Fungal Bloom in Australian Coastal Waters,Its Metabolites and Potential Impact on Symbiodinium Dinoflagellates

Dust has been widely recognised as an important source of nutrients in the marine environment and as a vector for transporting pathogenic microorganisms. Disturbingly, in the wake of a dust storm event along the eastern Australian coast line in 2009, the Continuous Plankton Recorder collected masses of fungal spores and mycelia (~150,000 spores/m³) forming a floating raft that covered a coastal area equivalent to 25 times the surface of England. Cultured A. sydowii strains exhibited varying metabolite profiles, but all produced sydonic acid, a chemotaxonomic marker for A. sydowii. The Australian marine fungal strains share major metabolites and display comparable metabolic diversity to Australian terrestrial strains and to strains pathogenic to Caribbean coral. Secondary colonisation of the rafts by other fungi, including strains of Cladosporium, Penicillium and other Aspergillus species with distinct secondary metabolite profiles, was also encountered. Our bioassays revealed that the dust-derived marine fungal extracts and known A. sydowii metabolites such as sydowic acid, sydowinol and sydowinin A adversely affect photophysiological performance (F_v/F_m) of the coral reef dinoflagellate endosymbiont Symbiodinium. Different Symbiodinium clades exhibited varying sensitivities, mimicking sensitivity to coral bleaching phenomena. The detection of such large amounts of A. sydowii following this dust storm event has potential implications for the health of coral environments such as the Great Barrier Reef.     

NaCl和Na2SO4胁迫对玉米幼苗渗透调节物质含量的影响

研究了NaCl和Na₂SO₄胁迫对玉米幼苗电解质渗漏、根系活力及脯氨酸、可溶性糖、可溶性蛋白等渗透调节物质含量的影响。结果表明，玉米在NaCl作用下其电解质渗漏、根系活力及渗透调节物质含量的变化和Na₂SO₄胁迫相似，电解质渗漏、根系活力及渗透调节物质含量均随着盐浓度的增加而加剧。NaCl胁迫下增幅大于Na₂SO₄胁迫，表明NaCl胁迫对玉米幼苗的伤害大于Na₂SO₄胁迫。     

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH₂), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 3₁₀-helix from residues Pro⁷ to His¹⁰ and a type I β-turn from residues Pro¹⁴ to Cys¹⁷. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC₅₀ of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu² by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser¹² with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.     

Saquayamycin B1 Suppresses Proliferation,Invasion, and Migration by Inhibiting PI3K/AKT Signaling Pathway in Human Colorectal Cancer Cells

Moromycin B (Mor B), saquayamycin B₁ (Saq B₁), saquayamycin B (Saq B), and landomycin N (Lan N), four angucyclines produced by the marine-derived actinomycete Streptomyces sp., are a class of polyketone compounds containing benzanthracene. Here, the structure–activity relationship of these four compounds was analyzed in human colorectal cancer (CRC) cells. Saq B₁, which showed the strongest cytotoxicity with an IC₅₀ of 0.18–0.84 µM for CRC cells in MTT assays, was employed to test underlying mechanisms of action in SW480 and SW620 cells (two invasive CRC cell lines). Our results showed that Saq B₁ inhibited CRC cell proliferation in a dose- and time-dependent manner. Notably, lower cytotoxicity was measured in normal human hepatocyte cells (QSG-7701). Furthermore, we observed proapoptosis, antimigration, and anti-invasion activities of Saq B₁ in CRC cells. At the same time, the protein and mRNA expression of important markers related to the epithelial–mesenchymal transition (EMT) and apoptosis changed, including N-cadherin, E-cadherin, and Bcl-2, in Saq B₁-treated CRC cells. Surprisingly, the PI3K/AKT signaling pathway was shown to be involved in Saq B₁-induced apoptosis, and in inhibiting invasion and migration. Computer docking models also suggested that Saq B₁ might bind to PI3Kα. Collectively, these results indicate that Saq B₁ effectively inhibited growth and decreased the motor ability of CRC cells by regulating the PI3K/AKT signaling pathway, which provides more possibilities for the development of drugs in the treatment of CRC.     

Ecklonia cava Attenuates PM2.5-Induced Cognitive Decline through Mitochondrial Activation and Anti-Inflammatory Effect

To evaluate the effects of Ecklonia cava (E. cava) on ambient-pollution-induced neurotoxicity, we used a mouse model exposed to particulate matter smaller than 2.5 µm in aerodynamic diameter (PM_2.5). The intake of water extract from E. cava (WEE) effectively prevented the learning and memory decline. After a behavioral test, the toll-like receptor (TLR)-4-initiated inflammatory response was confirmed by PM_2.5 exposure in the lung and brain tissues, and the WEE was regulated through the inhibition of nuclear factor-kappa B (NF-κB)/inflammasome formation signaling pathway and pro-inflammatory cytokines (IL-6 and IFN-γ). The WEE also effectively improved the PM_2.5-induced oxidative damage of the lungs and brain through the inhibition of malondialdehyde (MDA) production and the activation of mitochondrial activity (mitochondrial ROS content, mitochondria membrane potential (MMP), adenosine triphosphate (ATP) content, and mitochondria-mediated apoptotic molecules). In particular, the WEE regulated the cognition-related proteins (a decreased amyloid precursor protein (APP) and p-Tau, and an increased brain-derived neurotrophic factor (BDNF)) associated with PM_2.5-induced cognitive dysfunction. Additionally, the WEE prevented the inactivation of acetylcholine (ACh) synthesis and release as a neurotransmitter by regulating the acetylcholinesterase (AChE) activity, choline acetyltransferase (ChAT), and ACh receptor (AChR)-α3 in the brain tissue. The bioactive compounds of the WEE were detected as the polysaccharide (average Mw; 160.13 kDa) and phenolic compounds including 2′-phloroeckol.     

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells

Background:Bispecific antibodies represent an important class of mAbs, with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti- CD3 × CD19 bsAb is a bispecific T-cell engager (BiTE) currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods:WPRE sequence was incorporated at the 3’ end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion:Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in HEK-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody. Key Words: Acute lymphoblastic leukemia, Bispecific antibodies, Monoclonal antibody     

Gloeothece sp.—Exploiting a New Source of Antioxidant,Anti-Inflammatory,and Antitumor Agents

Bioactive lipidic compounds of microalgae, such as polyunsaturated fatty acids (PUFA) and carotenoids, can avoid or treat oxidation-associated conditions and diseases like inflammation or cancer. This study aimed to assess the bioactive potential of lipidic extracts obtained from Gloeothece sp.–using Generally Recognized as Safe (GRAS) solvents like ethanol, acetone, hexane:isopropanol (3:2) (HI) and ethyl lactate. The bioactive potential of extracts was assessed in terms of antioxidant (ABTS^•+, DPPH^•, ^•NO and O₂^•assays), anti-inflammatory (HRBC membrane stabilization and Cox-2 screening assay), and antitumor capacity (death by TUNEL, and anti-proliferative by BrdU incorporation assay in AGS cancer cells); while its composition was characterized in terms of carotenoids and fatty acids, by HPLC-DAD and GC-FID methods, respectively. Results revealed a chemopreventive potential of the HI extract owing to its ability to: (I) scavenge ^-NO^• radical (IC₅₀, 1258 ± 0.353 µg·mL⁻¹); (II) inhibit 50% of COX-2 expression at 130.2 ± 7.4 µg·mL⁻¹; (III) protect 61.6 ± 9.2% of lysosomes from heat damage, and (IV) induce AGS cell death by 4.2-fold and avoid its proliferation up to 40% in a concentration of 23.2 ± 1.9 µg·mL⁻¹. Hence, Gloeothece sp. extracts, namely HI, were revealed to have the potential to be used for nutraceutical purposes.     

A New Cytotoxicity Assay for Brevetoxins Using Fluorescence Microscopy

Brevetoxins are a family of ladder-framed polyether toxins produced during blooms of the marine dinoflagellate, Karenia brevis. Consumption of shellfish or finfish exposed to brevetoxins can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are believed to be due to the activation of voltage-sensitive sodium channels in cell membranes. The traditional cytotoxicity assay for detection of brevetoxins uses the Neuro-2A cell line, which must first be treated with the neurotoxins, ouabain and veratridine, in order to become sensitive to brevetoxins. In this study, we demonstrate several drawbacks of the Neuro-2A assay, which include variability for the EC₅₀ values for brevetoxin and non-linear triphasic dose response curves. Ouabain/veratridine-treated Neuro-2A cells do not show a typical sigmoidal dose response curve in response to brevetoxin, but rather, have a polynomial shaped curve, which makes calculating EC₅₀ values highly variable. We describe a new fluorescence live cell imaging model, which allows for accurate calculation of cytotoxicity via nuclear staining and additional measurement of other viability parameters depending on which aspect of the cell is stained. In addition, the SJCRH30 cell line shows promise as an alternative to Neuro-2A cells for testing brevetoxins without the need for ouabain and veratridine.     

Purification and Characterization of a Fucoidanase (FNase S) from a Marine Bacterium Sphingomonas paucimobilis PF-1

The Search for enzyme activities that efficiently degrade marine polysaccharides is becoming an increasingly important area for both structural analysis and production of lower-molecular weight oligosaccharides. In this study, an endo-acting fucoidanase that degrades Miyeokgui fucoidan (MF), a sulfated galactofucan isolated from the sporophyll (called Miyeokgui in Korean) of Undaria pinnatifida, into smaller-sized galactofuco-oligosaccharides (1000–4000 Da) was purified from a marine bacterium, Sphingomonas paucimobilis PF-1, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose column chromatography, and chromatofocusing. The specific activity of this enzyme was approximately 112-fold higher than that of the crude enzyme, and its molecular weight was approximately 130 kDa (FNase S), as determined by native gel electrophoresis and 130 (S1), 70 (S2) and 60 (S3) kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of FNase S were pH 6.0–7.0 and 40–45 °C, respectively. FNase S activity was enhanced by Mn²⁺ and Na⁺ (115.7% and 131.2%), but it was inhibited by Ca²⁺, K⁺, Ba²⁺, Cu²⁺ (96%, 83.7%, 84.3%, and 89.3%, respectively), each at 1 mM. The K_m, V_max and K_cat values of FNase S on MF were 1.7 mM, 0.62 mg·min⁻¹, and 0.38·S⁻¹, respectively. This enzyme could be a valuable tool for the structural analysis of fucoidans and production of bioactive fuco-oligosaccharides.     

Microwave-Assisted Extraction of Phlorotannins from Fucus vesiculosus

Microwave-assisted extraction (MAE) was carried out to maximize the extraction of phlorotannins from Fucus vesiculosus using a hydroethanolic mixture as a solvent, as an alternative to the conventional method with a hydroacetonic mixture. Optimal MAE conditions were set as ethanol concentration of 57% (v/v), temperature of 75 °C, and time of 5 min, which allowed a similar recovery of phlorotannins from the macroalgae compared to the conventional extraction. While the phlorotannins richness of the conventional extract was slightly superior to that of MAE (11.1 ± 1.3 vs. 9.8 ± 1.8 mg PGE/g DW_extract), both extracts presented identical phlorotannins constituents, which included, among others, tetrafucol, pentafucol, hexafucol, and heptafucol structures. In addition, MAE showed a moderate capacity to scavenge ABTS^•+ (IC₅₀ of 96.0 ± 3.4 µg/mL) and to inhibit the activity of xanthine oxidase (IC₅₀ of 23.1 ± 3.4 µg/mL) and a superior ability to control the activity of the key metabolic enzyme α-glucosidase compared to the pharmaceutical drug acarbose.