Shmuel Carmeli

IDENTIFICATION OF CYLINDROSPERMOPSIN IN APHANIZOMENON OVALISPORUM (CYANOPHYCEAE) ISOLATED FROM LAKE KINNERET, ISRAEL1

Aphanizomenon ovalisporum (Forti) was identified and isolated from Lake Kinneret upon its first appearance as a dominant bloom in late 1994. This cyanobacterial species, not previously known to be toxic, was evaluated by a commonly used mouse bioassay and was demonstrated to induce toxic symptoms that were distinguishable from the typical symptoms of the neurotoxins previously reported in Aphanizomenon flos‐aquae (L.) Ralfs. Mice died 5–24 h after crude extracts were injected intraperitoneally, and the LD50 value was estimated as 465 mg dry wt biomass · kg−1 mouse. A toxicity‐guided fractionation of the active extract indicated that the potent substance is polar an nature. The structure of the active compound was determined by its mass spectrometry and NMR data. The compound was found to be the sulfate‐guanidinium zwitterion, cylindrospermopsin, previously isolated from the cyanobacterium Cylindrospermopsis raciborskii (Woloszynska) and recently also reported in Umezakia natans (Watanabe). This is the first time that Aphanizomenon ovalisporum has been reported to contain a toxic compound.

Uracil moiety is required for toxicity of the cyanobacterial hepatotoxin cylindrospermopsin.

A new natural derivative of the sulfated guanidinium zwitterionic toxin cylindrospermopsin, 7-epi-cylindrospermopsin, was recently isolated from the cyanobacterium Aphanizomenon ovalisporum (Forti). The toxicity of the molecule (LD50 ip 5 d), estimated by mouse bioassay, was 200 µg/kg mouse, a value similar to that of cylindrospermopsin. Treatment of cylindrospermopsin with chlorine solution or chlorine-related oxidants produced two new derivatives. The chemical structure of these products was elucidated by nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques and toxicity was determined. In the first derivative, the vinylic proton at position 5 of the pyrimidine ring was substituted by chlorine to yield 5-chlorocylindrospermopsin. The other product is a truncated one, where C-6 of the pyrimidine ring was oxidized to a carboxylic acid. A trivial name, cylindrospermic acid, was given to this compound. Both products showed no toxic effects even at doses 50 times higher than the LD50 of cylindrospermopsin (10 mg/kg mouse ip). Based on these results, the pyrimidine ring is postulated as the molecule component essential for the toxicity of cylindrospermopsin.

Diversity and potential antifungal properties of fungi associated with a Mediterranean sponge

Fungi that inhabit marine sponges occupy an ecological niche that has recently attracted great attention due to the potential in either ecological or pharmaceutical advances. The ecological interaction between marine sponges and fungi is, however, only poorly understood. Eighty five fungal taxa were isolated from the marine sponge Psammocinia sp. from the Mediterranean Sea. The majority (89%) of these taxa were isolated using a `sample compressing` method, in combination with the use of fungicides-amended medium. Abundant `terrestrial` taxa such as Acremonium, Penicillium and Trichoderma were found along with potentially undescribed Phoma and Trichoderma species. Several of these taxa exhibited in vitro anti-fungal properties as determined against four test fungi. Even though a significant number of fungal taxa were isolated during this study, we estimate that the diversity of fungi that are associated with Psammocinia sp. is higher than reported here. It is advocated that Psammocinia, and other sponge genera, may be a prime niche for discovering new fungal species as well as novel anti-fungal compounds from fungal sources.

Inhibitors of serine proteases from a waterbloom of the cyanobacterium Microcystis sp.

Abstract Three new protease inhibitors, micropeptins SF909 (1) and SF995 (2) and microcin SF608 (3), were isolated from the hydrophilic extract of a microcystis sp. waterbloom. The planar structure of compounds 1–3 was determined by homonuclear and inverse-heteronuclear 2D-NMR techniques as well as high-resolution mass spectrometry. The absolute configuration of the asymmetric centers was studied using Marfeys method for HPLC. Micropeptin SF909 (1) inhibited chymotrypsin with IC50 of 4.0 μg/mL while micropeptin SF995 (2) and microcin SF608 (3) inhibited trypsin with IC50s of 0.2 and 0.5 μg/mL, respectively.

In vitro chemopreventive potential of fucophlorethols from the brown alga Fucus vesiculosus L. by anti-oxidant activity and inhibition of selected cytochrome P450 enzymes.

Within a project focusing on the chemopreventive potential of algal phenols, two phloroglucinol derivatives, belonging to the class of fucophlorethols, and the known fucotriphlorethol A were obtained from the ethanolic extract of the brown alga Fucus vesiculosus L. The compounds trifucodiphlorethol A and trifucotriphlorethol A are composed of six and seven units of phloroglucinol, respectively. The compounds were examined for their cancer chemopreventive potential, in comparison with the monomer phloroglucinol. Trifucodiphlorethol A, trifucotriphlorethol A as well as fucotriphlorethol A were identified as strong radical scavengers, with IC(50) values for scavenging of 1,1-diphenyl-2 picrylhydrazyl radicals (DPPH) in the range of 10.0-14.4 microg/ml. All three compounds potently scavenged peroxyl radicals in the oxygen radical absorbance capacity (ORAC) assay. In addition, the compounds were shown to inhibit cytochrome P450 1A activity with IC(50) values in the range of 17.9-33 microg/ml, and aromatase (Cyp19) activity with IC(50) values in the range of 1.2-5.6 microg/ml.

Protease inhibitors from a water bloom of the cyanobacterium Microcystis aeruginosa

Abstract Five new protease inhibitors, micropeptins SD944 ( 1 ), SD979 ( 2 ), SD999 ( 3 ) and SD1002 ( 4 ) and microginin SD755 ( 5 ) were isolated along with two known inhibitors, micropeptin SF995 ( 6 ) and microcin SF608 ( 7 ), from the hydrophilic extract of Microcystis aeruginosa . The planar structure of compounds 1 – 5 was determined by homonuclear and inverse-heteronuclear 2D-NMR techniques as well as high-resolution mass spectrometry. The absolute configuration of the asymmetric centers was studied using Marfeys method for HPLC. Compounds 1 – 4 , 6 and 7 are serine-protease inhibitors while compound 5 was found to inhibit amino-proteases.

Presence of Aspergillus sydowii, a pathogen of gorgonian sea fans in the marine sponge Spongia obscura.

The fungus Aspergillus sydowii is the causative agent of epidemics that affect gorgonian corals (sea fans) and has significantly affected their populations in the Caribbean Sea. We have isolated a strain of A. sydowii from healthy marine sponges (Spongia obscura) collected in Bahamian inshore waters. After its identification on the basis of morphology, molecular markers and chemical profiling followed by pathogenicity tests, we found this strain to be highly similar to a strain isolated from diseased coral, and have shown the capacity of this fungus to persist in sponge environment. Our findings suggest that sponges have the possibility of being reservoirs of a potential marine pathogen.

“Non-Toxic” Cyclic Peptides Induce Lysis of Cyanobacteria—An Effective Cell Population Density Control Mechanism in Cyanobacterial Blooms

The presence of planktopeptin BL1125, anabaenopeptin B and anabaenopeptin F, two types of “non-toxic” cyclic peptide produced in bloom forming cyanobacteria, can provoke lysis of different non-axenic Microcystis aeruginosa cell lines via the induction of virus-like particles. The resulting particles are also able to infect the axenic M. aeruginosa cell line without lytic effects. Nevertheless, the presence of “non-toxic” cyclic peptides of cyanobacterial origin can induce lysis of these previously infected cells. This effect implies that a possible role of these peptides in the natural environment is the control of cyanobacterial population density. Lysogenic cyanobacteria can consequently act as hot-spots that, in the presence of cyanobacterial cyclic peptides, release numerous infectious particles. The process can be self-augmented with the simultaneous release of additional cyclic peptides from the producing lysogens, starting a forest fire effect that ends in collapse of cyanobacterial blooms.

Umbelliferone, a phytoalexin associated with resistance of immature Marsh grapefruit to Penicillium digitatum

Abstract A low concentration (2 mg −1 FW) of 7-hydroxycoumarin (umbelliferone) was found in albedo of non-inoculated mature yellow Marsh grapefruit ( Citrus paradisi ). The concentration of umbelliferone in albedo of immature green grapefruit (2 months before full maturity) was 17 mg g −1 FW. Four days following inoculation with Penicillium digitatum , the accumulation of umbelliferone in the albedo of mature and immature grapefruit was 28 and 250 mg g −1 FW, respectively. By that period, the infected area in the yellow fruit was as large as 230 mm 2 , as compared with 8 mm 2 in the green fruit. The EC 50 of umbelliferone to P. digitatum growth in vitro was 95 mg ml −1 . Additionally, umbelliferone inhibited growth of several other pathogenic fungi in vitro . It is suggested that umbelliferone plays a role in the defense mechanisms of immature grapefruit against P. digitatum .

Modified peptides from a water bloom of the cyanobacterium Nostoc sp.

Abstract Six new metabilites, nostopeptin BN920 ( 1 ), nostoginin BN741 ( 2 ), nostoginin BN578 ( 3 ), banyascyclamide A ( 4 ) banyascyclamide B ( 5 ) and banyascyclamide C ( 6 ), were isolated from the hydrophilic extract of a Nostoc sp. The planar structure of compounds 1 – 6 was determined by homonuclear and inverse-heteronuclear 2D NMR techniques as well as high-resolution mass spectrometry. The absolute configuration of the asymmetric centers was studied using Marfeys method for HPLC. Compound 1 inhibits the serine-protease chymotrypsin while compound 2 inhibits amino-proteases.