The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C₉H₁₆N₆O₂. This structure differed from that of decarbamoyl STX (dcSTX; C₉H₁₆N₆O₃) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.     

Discovery of new low-molecular-weight p53–Mdmx disruptors and their anti-cancer activities

Although several p53–Mdm2-binding disruptors have been identified to date, few studies have been published on p53–Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200–300 selectively inhibited the p53–Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53–Mdmx interaction over the p53–Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53–Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.     

Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5·10⁻⁵ M in the culture medium. The protection afforded by anitoxidants was then tested: α-tocopherol gave at 10⁻⁴ M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Purification of prostaglandin E2-9-oxoreductase from human decidua vera

Prostaglandin E2-9- oxoreductase (PGE2-9-OR), the enzyme which converts prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been detected in human decidua vera. A 105-fold purification was achieved when the centrifuged homogenate was fractionated sequentially by DEAE-Trisacryl, hydroxyapatite-agarose gel, ultrogel AcA 44 and Matrex gel blue A gel chromatographies. The following kinetic constants for PGE2-9-OR have been obtained. The equilibrium constant with respect to PGE2 is 83 microM, the Michaelis constant, Km, for PGE2 is 80 microM, for NADPH 1.6 microM. The maximal velocity for the forward reaction is V1 = .203 pmol/min. The enzyme was inhibited by progesterone, oestradiol-17 beta, cortisol and pharmaceutical drugs. An activating effect could be demonstrated with Ca2+ and oxytocin. The occurrence of PGE2-9-OR in the decidua vera suggests that this enzyme may be responsible for the transformation of PGE2 to PGF2 alpha in these tissues. This may be an important mechanism for the initiation and maintenance of uterine contractions.     

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.     

CircRNA has_circ_0006427 suppresses the progression of lung adenocarcinoma by regulating miR-6783–3p/DKK1 axis and inactivating Wnt/β-catenin signaling pathway

Recently, circular RNAs (circRNAs) are identified as a novel class of noncoding RNAs playing important roles in human malignant tumors. However, the regulatory function of circRNA in lung adenocarcinoma (LUAD) is still largely unknown. Present study aimed to explore the role of circ_0006427 in LUAD progression. Firstly, the downregulation of circ_0006427 in LUAD tissues and cell lines was revealed by microarray analysis and qRT-PCR analysis. And we also confirmed the circ_0006427 as a prognostic target in LUAD patients. Functionally, overexpression of circ_0006427 effectively suppressed cell proliferation, migration and invasion. Mechanistically, circ_0006427 was found to be predominantly located in the cytoplasm of LUCA cell, and was further revealed to positively regulate DKK1 in LUAD by sponging miR-6783–3p. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and western blot analysis revealed that circ_0006427 inactivated Wnt/β-catenin signaling pathway by upregulating DKK1. At last, rescue assays proved the function of circ_0006427/miR-6783–3p/DKK1 axis in LUAD progression. In conclusion, our study revealed that circ_0006427 suppressed lung adenocarcinoma progression through regulating miR-6783–3p/DKK1 axis.     

Mode of action of a surfactant–polymer formulation to support performance of the entomopathogenic nematode Steinernema carpocapsae for control of diamondback moth larvae (Plutella xylostella)

The use of entomopathogenic nematodes on cabbage leaves against larvae of the diamondback moth (DBM) Plutella xylostella requires the addition of formulation adjuvants to achieve satisfying control. Without adjuvants nematodes settle in the tank mix of backpack sprayers causing uneven distribution. The polymers arabic and guar gum, alginate and xanthan were used in concentrations between 0.05 and 0.3% to retard sedimentation of Steinernema carpocapsae. Arabic gum had no effect, guar gum prevented sedimentation at 0.3% but the effect dropped significantly at lower concentration. At 0.05%, xanthan prevented nematode sedimentation better than alginate. Deposition of nematodes on the leaves was significantly increased by the addition of any of the polymers. Spraying nematodes on leaves with an inclination of 45° without the addition of any formulation resulted in 70% run-off. Adding 0.2% alginate or xanthan reduced the losses to <20%. The use of a surfactant–polymer formulation significantly reduced defoliation by DBM larvae. Visual examinations provided evidence that nematodes are not ingested by DBM larvae. Invasion of S. carpocapsae is an active process via the anus. The function of the formulation is not to prolong nematode survival, but to provide environmental conditions which enable rapid invasion of the nematodes. Nematode performance was improved by selection of the best surfactant in combination with xanthan and by optimisation of the concentrations of the surfactant Rimulgan® and the polymer xanthan. The best control results were achieved with Rimulgan® at 0.3% together with 0.3% xanthan, causing DBM mortality of >90% at 80% relative humidity and >70% at 60%. The formulation lowered the LC₅₀ from 12 to 1 nematode/larva. The viscosity of the surfactant–polymer formulations correlated well with nematode efficacy, prevention of sedimentation and adherence to the leave. This physical parameter can therefore be recommended for improvement of nematode formulations to be used for foliar application against DBM.     

Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.