Shellfish poisons.

In our ongoing search for bioactive metabolites from marine organisms, novel shellfish poisons have been isolated. Pinnatoxins, which are amphoteric polyether compounds, were purified from the Okinawan bivalve Pinna muricata. Pinnatoxins show acute toxicity against mice and activate Ca2+ channels. Two novel alkaloids, pinnamine and pinnaic acid, were also obtained from P. muricata. Pinnaic acid inhibits cytosolic phospholipase (cPLA2). Pteriatoxins, which are pinnatoxin analogs, were isolated from the Okinawan bivalve Pteria penguin. A nanomole-order structure determination of pteriatoxins was achieved by the detailed analysis of 2D-NMR and ESI-TOF MS/MS. This review covers the isolation, structure determination, bioactivity, synthesis, and biogenesis of these shellfish poisons and related compounds.

Structure-activity studies of the nonphorbol tumor promoter palytoxin in Swiss 3T3 cells.

Derivatives of palytoxin have been prepared which are modified on either the hydroxyl terminus or the amino terminus of the molecule. Previously we have shown that palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter, can inhibit epidermal growth factor binding in Swiss 3T3 cells through a pathway which is sodium dependent but not calcium or protein kinase C dependent. We used the epidermal growth factor receptor system to determine whether the specific chemical modifications of palytoxin present in these derivatives alter the cellular mechanism of action of the toxin. The dose response and ion dependence of palytoxin, the hydroxyl terminus derivative palytoxin-COOH, and the amino terminus derivatives N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were compared with respect to inhibition of epidermal growth factor binding. The potency of palytoxin-COOH was similar to that of palytoxin. By contrast, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were approximately 1/100 as potent as palytoxin in this assay. All three derivatives were at least 100-fold less toxic than palytoxin. Like palytoxin, the activities of palytoxin-COOH, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were dependent upon the presence of extracellular sodium. However, there was a significant difference in the dependence of the derivatives on extracellular calcium. Our results suggest that the hydroxyl terminus is important for determining the calcium dependence of the molecule and the amino terminus is important for determining the biological potency of palytoxin. We conclude that modification of the hydroxyl terminus region is an effective means of reducing the toxicity of palytoxin while retaining the biological effects.

Induction of topoisomerase I-mediated DNA cleavage by a new indolocarbazole, ED-110.

ED-110 is a new semisynthetic antitumor agent derived from a novel indolocarbazole antibiotic, BE-13793C, produced by an actinomycete. ED-110 induced topoisomerase I-mediated DNA cleavage in vitro as strongly as camptothecin, whereas topoisomerase II-mediated DNA cleavage was not induced by this agent. Exposure of P388 cells to ED-110 caused a typical topoisomerase toxicity, i.e.: formation of cleavable complexes; inhibition of nucleotide synthesis rather than protein synthesis; and cell cycle arrest in G2. ED-110 inhibited the growth of P388 cells, with a 50% growth-inhibitory concentration of 44 nM. ED-110 is distinguished from camptothecin by its very different structure and its ability to intercalate into double-stranded DNA. These results suggest that ED-110 has potential as a novel antitumor agent targeting topoisomerase I.

N-Acetylgalactosamine 4,6-bissulfate in rat urine. I. Isolation, identification and chemical synthesis.

By starting with 4 1 of rat urine, it was possible to obtain a sulfate ester of hexosamine in crystalline form. A series of identification procedures including chemical analyses, enzymatic digestion, proton magnetic resonance spectroscopy and infrared spectroscopy showed that this substance is 2-acetamido-2-deoxy-D-galactose 4,6-bissulfate. The trivial name for this compound is N-acetylgalactosamine 4,6-bissulfate; Quantitation by isotopic techniques indicated the urine possessed an average concentration of 8 muM N-acetylgalactosamine 4,6-bissulfate. Further extension of these studies necessitated the chemical synthesis of N-acetylgalactosamine 4,6-bissulfate and related compounds to be used for references or as biological substrates. Direct sulfation of N-acetylgalactosamine was attempted first, and strong preference for attack on the primary hydroxyl group (position 6) was found for chlorosulfonic acid. Thus, the reaction with 2.2 molar equivalents of the sulfating agent gave N-acetylgalactosamine 6-sulfate and its derivatives bearing a second sulfate at either position 1 (minor) or position 3 (major). The lack of sulfation at position 4 could be attributed to steric effects of the sulfate group preferentially attached to position 6. Another experiment in which UDP-N-acetylgalactosamine 4-sulfate was used in place of the free sugar led to the formation of a bissulfated sugar-nucleotide which, on subsequent hydrolysis with mild acid, afforded N-acetylgalactosamine 4,6-bissulfate, the same compound as that obtained from rat urine.

Single ionic channels induced by palytoxin in guinea-pig ventricular myocytes.

1. Mechanisms of palytoxin-induced ion permeability were examined in isolated single ventricular cells of guinea-pig under whole-cell-attached patch clamp conditions. 2. Palytoxin (1-2 x 10(-11) M, dissolved in Tyrode solution and put in the patch electrode) induced an elementary current flowing through single channels. Direction of the current was inward and the amplitude was 0.65 +/- 0.03 pA (mean +/- s.e. mean) at the resting membrane potential. The amplitude increased linearly with membrane hyperpolarization and decreased with depolarization; the single channel conductance was 9.5 +/- 0.5 pS. 3. Palytoxin-induced single channel current was resistant to tetrodotoxin (5 x 10(-5) M) or cobalt ions (2 x 10(-3) M) and was observed under Ca-free conditions. However, no channel current was induced by palytoxin (10(-11) - 10(-9) M) dissolved in Na+-free, choline-Tyrode solution. 4. Palytoxin also induced single channel currents in Na+-free, NH4+-, Li+- or Cs+-Tyrode solution, and the slope conductances were 16.5 +/- 1.6 pS, 9.2 +/- 0.7 pS and 11.0 +/- 0.7 pS, respectively. 5. These results indicate that palytoxin forms a new type of ionic channel with unique ion selectivity and gating behaviour.

Inhibitory effect of a toxin okadaic acid, isolated from the black sponge on smooth muscle and platelets.

1. Effects of okadaic acid, a toxin isolated from marine sponges, on smooth muscle contraction and platelet activation were examined. 2. Contractions in rabbit aorta induced by high concentrations of K+ and noradrenaline were inhibited by 0.1-1 microM okadaic acid in a concentration-dependent manner. Spontaneous rhythmic contractions as well as high K+-induced contraction in guinea-pig taenia caeci were also inhibited by 1 microM okadaic acid. 3. High K+-induced contraction in rabbit aorta was accompanied by increased Ca2+ influx measured with 45Ca2+ and increased cytosolic Ca2+ [( Ca2+]cyt) measured with fura-2-Ca2+ fluorescence. Okadaic acid inhibited the contraction without inhibiting Ca2+ influx and produced only a small decrease in [Ca2+]cyt. 4. In a saponin-skinned taenia, Ca2+-induced contraction was not inhibited but rather potentiated by okadaic acid. 5. Okadaic acid, 1 microM, inhibited aggregation, ATP release and increased in [Ca2+]cyt induced by thrombin in washed rabbit platelets. Okadaic acid itself did not change the platelet activities. 6. Okadaic acid did not change the cyclic AMP content of rabbit aorta although the inhibitory effects of okadaic acid were similar to those of cyclic AMP. 7. Although the mechanism of the inhibitory effect of okadaic acid was not clarified in the present experiments, it is suggested that okadaic acid acts by inhibiting protein phosphatases resulting in an indirect activation of cyclic AMP-dependent protein phosphorylation.

Increase in potential activities of protein phosphatases PP1 and PP2A in lymphoid tissues of autoimmune MRL/MpJ-lpr/lpr mice.

The differential assay conditions for protein phosphatases PP1, PP2A, and PP2C were extensively studied by using crude extracts from mouse lymphoid tissues as enzyme sources. Under these conditions, the protein phosphatase activities were measured in MRL/MpJ-lpr/lpr mice (MRL/lpr mice), autoimmune-prone mice, and MRL/MpJ(-)+/+ mice (MRL/+/+ mice) and C3H/HeJ mice as the controls. In MRL/lpr mice, significant alterations in protein phosphatase activities from those in the control mice were demonstrated. In spleen and liver from MRL/lpr mice, potential activities of PP1 and PP2A were distinctly elevated over those of the control mice. These elevations appeared to be due to accumulation of the abnormal lymphocytes that emerged in MRL/lpr mice. Although the PP1 activity in MRL/lpr lymph nodes was lower than those of normal spleen and thymus, it was greatly increased by Co(2+)-trypsin treatment so that the PP1 activity of MRL/lpr lymph nodes was the highest among those of all the tissues examined. In contrast, PP2C activity showed no remarkable alteration in the autoimmune disease model mice as compared with that in the control mice. These results demonstrated a specific elevation in potency of protein dephosphorylation in the tissues of MRL/lpr mice, suggesting a new explanation for the defect in signal transduction in this disease.

Enantioselective synthesis of attenols A and B.

[reaction: see text] Enantioselective synthesis of attenols A and B was accomplished by using diastereoselective hydroboration, Lindlar reduction, and acid-catalyzed acetal formation.

Endothelium inhibits the palytoxin-induced depolarization and Ca2+ mobilization in porcine coronary artery through endothelium-derived hyperpolarizing factor and nitric oxide released by palytoxin.

Palytoxin induced increases in cytosolic Ca2+ and tension, which were dependent on external Ca2+, and depolarized the membrane in endothelium-denuded porcine coronary arteries. When the endothelium was present, however, these effects were greatly inhibited, suggesting that some factors from endothelium inhibited the palytoxin-actions. Pretreatment with 100 microM N omega-nitro-L-arginine partially reversed the inhibitory effect of endothelium on the Ca2+ movement and the contraction but not that on the depolarization. Pretreatment with 10 microM indomethacin did not affect the inhibition. These results suggest that palytoxin released both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) from the endothelium, both of which counteracted the actions of palytoxin on smooth muscle cells. It is thought that the palytoxin-induced depolarization was attenuated by hyperpolarization due to EDHF.

A new topoisomerase II inhibitor, BE-22179, produced by a streptomycete. I. Producing strain, fermentation, isolation and biological activity.

A new topoisomerase II inhibitor, designated BE-22179, was isolated from the culture broth of Streptomyces sp. A22179, which resembles "Streptomyces gangtokensis". The inhibitor was extracted from the mycelial cake of the culture broth with organic solvent and successively purified by silica gel chromatography. BE-22179 inhibited topoisomerase II strongly but not topoisomerase I and showed potent antitumor activity against various tumor cell lines both in vitro and in vivo.

Isolation and structure of korolkoside, a bis-iridoid glucoside from Lonicera korolkovii.

A new bis-iridoid glucoside, korolkoside (1), was isolated from Lonicera korolkovii, and its structure and stereochemistry were determined by spectroscopic analysis and chemical derivatization. Korolkoside (1) consists of two secologanin moieties that are connected by an acetal linkage.

Possible mechanism of palytoxin-induced Ca++ mobilization in porcine coronary artery.

We investigated the mechanisms involved in palytoxin (PTX)-induced cytosolic Ca++ ([Ca++]i) mobilization and contraction in porcine coronary arteries using a fluorescent Ca++ indicator fura-PE3. PTX (1 pM-10 nM) induced concentration-dependent and sustained increases in [Ca++]i and tension, both of which were partially inhibited by 10 microM verapamil or 1 microM nicardipine. In Ca++-free solution containing 1 mM EGTA, PTX did not increase [Ca++]i. In nominally Ca++-free solution (no EGTA), however, PTX increased [Ca++]i, which was presumed to be due to release of Ca++ from intracellular stores. PTX-induced rise in [Ca++]i was dependent on external Na+ because it did not increase [Ca++]i in Na+-free solutions containing verapamil. An increase in [Ca++]i in response to 65.4 mM KCl also involved a verapamil-resistant but external Na+-dependent component. After blockage of voltage-dependent Ca++ channels with verapamil, elevation of external K+ to 65.4 mM enhanced the responses of [Ca++]i and tension to PTX. PTX at 10 and 100 pM depolarized the membrane by 4.5 +/- 0.8 and 18.6 +/- 1.7 mV, respectively. Because PTX is known to increase membrane Na+ permeability, our results suggest that an increase in cytosolic Na+ and the depolarization were primary events required for the PTX-induced Ca++ mobilization and that Ca++ influxes through voltage-dependent Ca++ channels and Na+-Ca++ exchange and Ca++ release from Ca++ stores, which was triggered by increased Ca++ entry, were responsible for the PTX-induced increase in [Ca++]i.

Cyclooxygenase induction is essential for NGF synthesis enhancement by NGF inducers in L-M cells.

Nerve growth factor (NGF) inducers, for example, 4-methylcatechol, pyrroloquinoline quinone, kansuinin A, and ingenol triacetate, stimulate NGF synthesis in L-M cells, but the mechanism of NGF induction by NGF inducers is not known. Using the four different types of previously described NGF inducers, we proved induction of cyclooxygenase activity by NGF inducers and detected prostaglandins D2 and E2 as metabolites of arachidonic acid. From the observation that the induction of NGF by each NGF inducers was inhibited by cyclooxygenase inhibitors or dexamethasone, cyclooxygenase activation is supposed to be an essential process for NGF induction.

Characteristics of palytoxin-induced depolarization in squid axons.

The effects of palytoxin on squid axon membranes have been studied by means of internal perfusion and voltage clamp techniques. When applied externally at a concentration ranging from 1 X 10(-8) to 1 X 10(-6) M, palytoxin caused a large depolarization of the membrane that was reversed slowly after washing with toxin-free solution. The toxin-induced depolarization disappeared quickly upon decreasing the external sodium concentration from 445 to 1 mM and the membrane was hyperpolarized beyond the original level. Palytoxin did not cause depolarization if both external and internal solutions were devoid of sodium. The toxin-induced depolarization was only reversed partially by external application of 1 X 10(-6) M tetrodotoxin. Palytoxin had no effect on the resting potential when perfused internally. These results indicate that palytoxin acts from outside of the nerve membrane to increase sodium permeability, thereby causing a depolarization. The palytoxin-poisoned membrane was also permeable to other cations with a permeability ratio of Na/Li/Cs/NH4 = 1:0.62:0.75:1.45. Under voltage clamp conditions, palytoxin shifted the voltage dependence of peak sodium current and steady-state potassium current in the direction of hyperpolarization and reduced markedly the maximum amplitude of peak sodium current. However, the kinetics of sodium current underwent little or no change except a shift of voltage dependence toward hyperpolarization. Leakage current increased markedly after application of palytoxin. In view of these observations and data from the literature, it was suggested that palytoxin creates a new channel in the membrane, thereby causing an increase in cation permeability, depolarization, and other effects such as muscle contraction.

Suppressive effect of norzoanthamine hydrochloride on experimental osteoporosis in ovariectomized mice.

Norzoanthamine is an alkaloid isolated from a colonial zoanthid. We examined the effects of norzoanthamine hydrochloride on bone weight, strength and morphology in ovariectomized mice, a postmenopausal osteoporosis model. Norzoanthamine hydrochloride significantly suppressed the decrease in femoral weight and bone biomechanical parameters caused by ovariectomy without an increase in uterine weight. This means that norzoanthamine hydrochloride does not have an estrogen-like effect on reproductive organs. Morphological observations of longitudinally ground sections of the humeri showed that norzoanthamine hydrochloride administration (2 mg/kg/d, p.o.) completely suppressed the loss of trabecular bone. Furthermore, norzoanthamine hydrochloride thickened the cortical bone. Based on these results, norzoanthamine hydrochloride may act as both a suppressor of bone resorption and an enhancer of bone formation in vivo.

Isolation and structures of haterumadioxins A and B, cytotoxic endoperoxides from the Okinawan sponge Plakortis lita.

Cytotoxic endoperoxides, haterumadioxins A (1) and B (2), were isolated from the Okinawan sponge Plakortis lita. Their structures were determined by spectroscopic analysis. The absolute stereostructure of 1 was determined by degradation reactions and the modified Mosher's method. Haterumadioxins showed significant cytotoxicity against 38 human cancer cell lines.

Isolation and structures of hedaols A, B, and C, new bisnorditerpenes from a Japanese brown alga.

New bisnorditerpenes, hedaols A (1), B (2), and C (3), were isolated from the Japanese brown alga Sargassum sp. Their structures were determined by spectroscopic analysis. The absolute stereocenter of 1 was determined by the modified Mosher's method. Hedaols showed low cytotoxicity against P388 cells.

Haterumaimides F-I, four new cytotoxic diterpene alkaloids from an ascidian Lissoclinum species.

Four new monochlorinated diterpene alkaloids, haterumaimides F-I (1-4), and two known ones, dichlorolissoclimide and chlorolissoclimide, were isolated from an ascidian Lissoclinum sp. Their structures with absolute stereochemistries were elucidated by chemical and spectral analyses. Haterumaimides F-I (1-4) inhibited the first cleavage of fertilized sea urchin eggs and exhibited potent to weak cytotoxicities against P388 cells.