An improved synthesis of (−)-brevisamide a marine cyclic ether isolated from the red-tide dinoflagellate Karenia brevis was achieved. The ether ring portion was constructed from an unsaturated lactone, which was prepared enantioselectively via an Evans aldol reaction and one-pot lactonization in the presence of excessive base after an Ando reaction. The ether ring and a dienol side chain fragment were connected via Suzuki–Miyaura coupling.

There has been little information demonstrating the roles of dimethylarginine dimethylaminohydrolase (DDAH), which is the hydrolyzing enzyme of endogenous nitric oxide synthase (NOS) inhibitors and, in turn, modulates the intracellular concentrations of NOS inhibitors, in the myometrium during the course of pregnancy. Therefore, the present experiments were designed to investigate whether or not DDAH activity, protein and mRNA expression levels are altered during gestation of the rat and, if altered, those changes reflect on the levels of endogenous inhibitors and endothelin-1 (ET-1), and NO-dependent cyclic GMP generation in the myometrium. The up-regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression at mid-gestation were accompanied by the reduced monomethylarginine and asymmetric dimethylarginine as NOS inhibitors, and ET-1 levels, and by the enhanced NO-dependent cyclic GMP production. At term gestation, on the other hand, down-regulated changes in DDAH activity, DDAH-2 protein and DDAH-2 mRNA expression were accompanied by the increased NOS inhibitors and ET-1 levels, and decreased NO-dependent cyclic GMP generation. These results suggest that alterations in DDAH/NOS inhibitors/NO-dependent cyclic GMP/ET-1 pathway are possibly involved in maintaining myometrial quiescence during gestation and controlling delivery at term.

Palytoxin (PLT) first isolated from zoanthids is extremely lethal to animals by intraperitoneal or intravenous administration but shows little toxicity by gavage dosing in contradiction to the occurrence of fatal poisoning due to PLT-containing seafood.In order to fully elucidate its potential risks to human we evaluated the toxicological effects via three ways of dosing: gavage, intra-tracheal administration (IT) and sublingual administration. A new analog, 42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin isolated from the dinoflagellate Ostreopsis siamensis and named ostreocin-D (OSD), was also used for comparison, additionally conducted by i.p. By gavage dosing, both toxins did not produce death in mice at the maximum dosage of 200 mu g/kg of PLT and 300 mu g/kg of OSD. Addition of dietary lipid components to PLT solutions for gavage or use of ulcerated mice did not alter the results, indicating no enhancement of PLT absorption. The two toxins were most toxic by the IT route, causing bleeding and alveolar destruction in the lung and resultant death at 2 mu g/kg of PLT, and 11 mu g/kg of OSD. Both toxins also induced organ injuries after 24 h when dosed by sublingual administration at about 200 mu g/kg. The injuries became fatal when PLT was dosed 2 or 3 times. The results pointed to the necessity of taking multiple approaches to assess the potential health risks due to PIT and its analogs in food and environments. (C) 2009 Elsevier Ltd. All rights reserved.

In a previous study, it was suggested that beta-cyclocitral plays an important role in understanding the lysis of cyanobacteria under natural conditions. The present study was conducted in order to understand how the beta-cyclocitral lyses cyanobacterial cells, and other anti cyanobacterial agents were also investigated. The preliminary study using a scanning electron microscope (SEM) demonstrated that there were three types of morphological changes in the cyanobacterial cells when they were incubated with anticyanobacterial agents: volatile compounds from cyanobacteria cause shrinking and then wrinkling, terpenoids contact directly and cause stripping; basic amino acids cause swelling and then collapsing. In order to clarify the extreme difference in the damage during the morphological changes between the beta-cyclocitral and L-lysine (Lys), the transmission electron microscope (TEM) technique was applied. Although a definite difference in the morphological damage was observed, a plausible mechanism for beta-cyclocitral could be not deduced. Throughout the experiments using antibiotics, it was found that the apparent morphological changes after lysis did not always correspond to the mode of action.

One of bacteria collected from Lake Sagami, Japan, Brevibacillus sp., was found to have a lytic activity of cyanobacteria, but did not produce active compounds. Instead, the co-culturing of Microcystis with the Brevibacillus sp. enhanced the production of two volatile compounds, beta-cyclocitral and 3-methyl-1-butanol, and the former had a characteristic lytic activity. It was confirmed that these volatile compounds were derived from the cyanobacteria themselves. beta-Ionone, geosmin and 2-methylisoborneol derived from cyanobacteria and similar volatile compounds, terpenoids, produced by plants also had a lytic activity. The minimum inhibitory concentration values of the cyanobacterial metabolites were estimated to be higher than those of compounds from plants except for a few compounds. Among them, beta-cyclocitral only produced a characteristic color change of culture broth from green to blue. This color change is similar to the phenomenon observed when a sudden decline in growth of cyanobacteria begins in a natural environment.

Diarrhetic activity of pectenotoxin-6 (PTX6), a shellfish contaminant in Japanese scallops (Patinopecten yessoensis), was studied in vivo. Mice gavaged with 5mg/kg PTX6 did not show diarrhea or fluid secretion, and no prominent pathological changes were observed. There was no synergistic toxicity of PTX6 with okadaic acid (OA) or pectenotoxin-2 (PTX2) when toxins were given to mice by gavage. Synergistic activity of PTX6 with OA was also not confirmed under crude conditional simulation with oil. In contrast to the oral administration to mice, PTX6 at 500μg/kg by i.p. was the lethal dose with bleeding in the liver, injuries at the gastric organs and the kidney. When rats were gavaged with PTX6 at a dose of 2mg/kg, PTX6 did not have diarrhetic activity; however, the middle–lower small intestine (jejunum–ileum) was eroded at villi by edema. PTX6 is a potent toxin if administered by intraperitoneal injection to mice, or if administered orally to the rat. However, it is not clear if PTX6 passes through the intestinal barrier if given by the oral route.

Surface topography has vital roles in cellular response. Here, to investigate the mechanism behind cellular response to surface topography, we prepared honeycomb (HC)-patterned films from poly(epsilon-caprolactone) (PCL) with micropatterned surface topography by casting a polymer solution of water-immiscible solvent under high humidity. We characterized the adsorption of fibronectin (Fn) on the film using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). The response of porcine aortic endothelial cells (PAECs) to adsorbed Fn molecules onto HC-patterned films was observed by immunofluorescence labeling of vinculin and the actin fiber of PAECs cultured for 1 and 72 h in serum-free medium. The expression of focal adhesion kinase autophosphorylated at the tyrosine residue (pFAK) at 1 h culture was determined using an immunoprecipitation method. Fn adsorbed selectively around the pore edges to form ring-shaped aggregates. The immunostaining results revealed that PAECs adhered to the HC-patterned films at focal contact points localized around pore peripheries. These points correspond to adsorption sites of Fn. The expression of pFAK after 1 h on the HC-patterned film was 3 times higher than that on a corresponding flat film, indicating that the signaling mediated by the binding between Fn and the integrin receptor was more highly activated on the HC-patterned film. These results suggest that the cellular response to HC-patterned films (e.g., adhesion pattern and phosphorylation of FAK) originates from the regularly aligned adsorption pattern of Fn determined by the pore structure of the film.

OBJECTIVE:To compare the effects of 10% dextran and epinephrine on intraosseous injection with lidocaine in rabbits. STUDY DESIGN:Twenty male Japanese white rabbits were used. The effect of intraosseous injection was evaluated using an electromyogram (EMG) of the digastric muscle after electrical pulp stimulation. Two percent lidocaine alone (L), 2% lidocaine containing 1:80000 epinephrine (LE8), 2% lidocaine containing 1:160 000 epinephrine (LE16), and 2% lidocaine containing 10% dextran (LD) were tested. Electromyogram recordings were repeated before and 30 seconds, 1, 2, 3, 4, 5, 7, 10, 12, 15, and 20 minutes after the intraosseous injection. Thereafter, recordings were repeated every 5 minutes until the EMG recovered to the control value. RESULTS:There was no difference in the onset time between the 4 groups. The order of the duration of maximum effect was LE8 >LE16 = LD >or=L. The order of the duration of anesthesia was LE8 >LE16 = LD >L. CONCLUSION:Ten percent dextran potentiates local anesthetic effects of 2% lidocaine in intraosseous injection. The potency of 10% dextran is comparable to 1:160 000 epinephrine.

Photophysical properties of meridional (mer) and facial (fac) isomers of eight iridium(III) trischelete complexes composed of combination of two ligands, 2-(p-tolyl)pyridine and 2-phenylpyrazole, were investigated. Characteristics of heteroleptic complexes were compared to the homoleptic complexes. Phosphorescence radiation process is rationally explained by inter-ligand energy transfer, however, mer → fac isomerization showed different behavior indicating participation of another excited state.

Synthetic azaspiracid-1 (AZA-1, 1), 6-, 10-, 13-, 14-, 16-, 17-, 19-, 20-epi-azaspiracid-1 (C1–C20-epi-AZA-1, 2), and twelve truncated azaspiracid-1 analogs (3–14) were synthesized and tested for their toxicity effects in mice. Of these compounds only AZA-1 (1) and its diastereomer C1–C20-epi-AZA-1 (2) exhibited significant toxicity in mice with the latter compound (2) being one-fourth as toxic as the former (1). The lack of toxicity exhibited by the severely truncated analogs (3–14) implies that the entire or at least a major part of the structure of AZA-1 (1) is required for biological activity.

A vibrating dental local anesthesia attachment (Vibraject, LLC, Calif) based on the concept of the gate-control theory has been used in clinical practice. The theoretical advantage of the vibrating needle is that it reduces the injection pain. We evaluated the effectiveness of Vibraject in combination with an electrical injection device. Injections were given into the alveolar mucosa adjacent to the root apex of the maxillary lateral incisor in 10 volunteers. Vibraject was randomly applied to either the left or right side of the injection. No statistically significant decrease in pain scores was found at needle insertion or anesthetic injection. The clinical efficacy of Vibraject remains controversial.

The intact Adda was isolated from microcystin-LR by a microbial degradation using an isolated Sphingomonas strain, B-9. The reaction of microcystin-LR with cell extract of this strain proceeded smoothly to give the final degradation product by way of two intermediates, linearized microcystin-LR and a tetrapeptide. The purified Adda that was structurally characterized using various spectral data did not show the toxicity to mice or inhibition to protein phosphatase activity in contrast to the native toxin.

Gambierol was isolated from Gambierdiscus toxicus, which causes ciguatera fish poisoning. The acute toxicological effects induced in mice by synthesized gambierol were studied. The lethal doses were about 80 microg/kg by i.p. and i.v., and 150 microg/kg by p.o. The main injury by this toxin was observed in the lung, and secondary in the heart, resulting in systemic congestion. Another toxic effect was seen in the stomach, inducing hypersecretion and ulceration. With survival from the severe stage during the initial 3 h, recovery was favorable, especially after 4 days. Additional effects were not evident during 1-week post-administration observation.

Toxicological effects of orally administered azaspiracid (AZA), a new toxin isolated from mussels, were investigated. First, a total of 25 mice were administered AZA twice at 300-450 microg/kg doses and observed for recovery processes from severe injuries. Slow recoveries from injuries were revealed: erosion and shortened villi persisted in the stomach and small intestine for more than 3 months: edema, bleeding, and infiltration of cells in the alveolar wall of the lung for 56 days; fatty changes in the liver for 20 days; and necrosis of lymphocytes in the thymus and spleen for 10 days. Secondly, low doses of AZA (50, 20, 5 and 1 microg/kg) were administered twice a week up to 40 times to four groups of mice. Many mice, nine out of ten at 50 microg/kg and three out of ten at 20 microg/kg, became so weak that they were sacrificed before completion of 40 injections. All these mice showed interstitial pneumonia and shortened small intestinal villi. Most importantly, lung tumor were observed in four mice, one out of ten (10%) at 50 microg/kg and three out of ten (30%) at 20 microg/kg. Tumors were not observed in 11 mice treated at lower doses and in 19 control mice. Hyperplasia of epithelial cells was also observed in the stomach of six mice out of ten administered at 20 microg/kg.

Two metabolites of microcystin-LR glutathione conjugate and, microcystin–cysteine conjugate, as well as microcystin-RR (MCRR) are less toxic than microcystin-LR (MCLR). In the present study, we investigated why these compounds are weakly toxic in comparison with MCLR, as the reason is still unknown and no systematic study has so far been carried out for a clarification of this issue. Although they showed almost the same inhibitory activity against protein phosphatases 1 and 2A as MCLR in vitro, the apparent toxicity of these three compounds by intratracheal administration to mice decreased to about 1/12 the level of MCLR at 100 μg/kg. An immunostaining study showed that these conjugates at a sublethal dose of 200 μg/kg were prominently observed in the intestine and kidney, whereas effective accumulation and bleeding were not found in the liver in spite of the larger dosage. As an explanation for these results, there may be two possibilities. First, the transport system to the liver might not function well, and second, transported toxins may be effectively eliminated by an appropriate system such as the GS-X (ATP-dependent glutathione S-conjugate exported) pump. It was concluded that the inhibitory activity against protein phosphatases is not always related to the apparent LD50 level, and that the appearance of toxicity by microcystins depends on the balance between accumulation and metabolism in the liver.

This paper describes the distribution and excretion of okadaic acid (OA) administered orally to mice and examined by immunostaining method. Five min after administration, OA was systemically distributed, being detected in the lung, liver, heart, kidney, and small and large intestine. The swollen small intestinal villi contained OA, causing the separation of epithelial cells from villi and erosion, which developed within 1h. Bleeding and edema in the lung were also found, and the distribution of OA coincided with these injuries. Although a considerable amount of OA was accumulated in the liver, no symptoms, such as bleeding, were observed. The detection of OA continued for 2 weeks in the liver and blood vessels. Excretion from kidney, cecum and large intestine began even after 5min of the administration, and the excretion from intestine continued for 4 weeks.