Hemolytic Compounds Research Articles

Assessment of Carcinogenic/Mutagenic Potential of Different Series of Synthetic Compounds

The new drug research is usually based on synthesis medicine. The use of these medications has created problems such as tolerance in humans, for a long time and due to legitimate use of anti-infection, microbial defense against branded medication is growing. A mutagensis study by Ames in the early 1970's, used worldwide by drug and chemicals companies to diagnose mutagens carcinogenes, making it possible for them to be detected, and to be added to the mutagenic synthesis portion or radiation source triggering irreversible changes, and to the genetic material transmitted from the parent. deoxyribonucleic acid (DNA). The purpose of this study was to assessment of carcinogenicity of synthetic compounds series by hemolytic, Ames and Damaged DNA protection assay. The cytotoxicity was determined with hemolytic assay and DNA Damage protection assay while mutagenicity was resolute by using S. typhimurium TA100 and TA98 strains. It is concluded that the compounds with less hemolytic compounds are good for uses in drugs. Synthetic compounds were determined to be non- mutagenic in nature. Analysis of variance (ANOVA) was applied to compare the hemolysis percent between different concentrations.

Peptide Toxins as Biothreats and the Potential for AI Systems to Enhance Biosecurity.

Biological weapons have been used for thousands of years, but recent advances in synthesis technologies have made peptide and protein toxin production more accessible and pose a threat to biosecurity worldwide. Natural toxins such as conotoxins, certain hemolytic compounds, and enterotoxins are peptide agents that can be synthesized in an environment with weak biosecurity measures and rudimentarily weaponized for limited use against smaller targets for lethal or nonlethal effects. Technological advances are changing the threat landscape around biological weapons and potentially facilitating a shift from state sponsored to more micro-level threats stemming from terror cells, insider threats, and lone wolf attacks. Here, we present the reader with an overview of the threat of peptide and protein toxins, provide examples of potent peptide toxins, and introduce capabilities of a proposed biosecurity program utilizing artificial intelligence that unifies commercial nucleotide and peptide synthesis vendors.

Hemolytic compound 3,7,11,15-tetramethyl-hexadecan-1,19-disulfate found in the invasive European sea squirt Ascidiella aspersa

The solitary European sea squirt Ascidiella aspersa was introduced to northern Japan, causing dense coverage of the aquacultured Japanese scallop Mizuhopecten yessoensis. There are concerns that allelopathy by the sea squirt will suppress the growth of the scallop. Herein, we studied the allelopathic and defensive potential of A. aspersa using hemolytic assay. An alkyl sulfate, viz. 3,7,11,15-tetramethyl-hexadecan-1,19-disulfate (1), was isolated as a hemolytic compound, and 1 showed mild toxicity against the Japanese scallop and brine shrimp.

Live Cell Microscopy and Flow Cytometry to Study Streptolysin S-Mediated Erythrocyte Hemolysis.

The ability to induce hemolysis, the rupturing of erythrocytes with the consequent release of their intracellular contents, is a phenotypic hallmark of a number of microbial toxins. Streptococcus pyogenes or Group A Streptococcus (GAS) is a human pathogen responsible for a wide range of diseases from mild pharyngitis to severe conditions such as toxic shock syndrome. GAS produces a powerful hemolytic toxin called streptolysin S (SLS). Herein, we describe a procedure for the preparation of SLS toxin and the use of two complementary approaches, live microscopy and flow cytometry, to study the effects of the SLS toxin on erythrocytes. In addition to providing insights into SLS-mediated hemolysis, these assays have the potential to be modified for the study of other hemolytic toxins and compounds.

Structural analyses of a hemolytic compound found in an extract of Hypsizygus marmoreus fruiting bodies at a low pH.

The current study determined the structure of a hemolytic compound found in an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus when its pH was lowered. The hemolytic compound was purified using the modified Bligh and Dyer method followed by chromatography using reversed phase and silica gel columns. Structural analyses of the purified hemolytic compound were performed using NMR and ESI-MS. The deduced structure indicated a trans,trans-5,8-docosadienoic acid calcium salt. Although numerous proteinous hemolysins from various mushrooms have been described, the current study is the first to report on a low-molecular-weight hemolytic compound derived from an H. marmoreus extract.

PH-Dependent exhibition of hemolytic activity by an extract of Hypsizygus marmoreus fruiting bodies.

The current study found that an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus exhibited hemolytic activity against sheep red blood cells when its pH was lowered. Although hemolytic activity was not detected when an extract had a neutral pH, an extract with a low pH exhibited potent hemolytic activity. The maximal hemolytic activity was exhibited by an extract with a pH of 5.5. A heat-treated extract did not exhibit hemolytic activity before its pH was lowered, and that activity was inhibited in the presence of PMSF and EDTA. The turbidity of the extract increased during lowering of its pH, and the precipitate fraction exhibited hemolytic activity. Fractionation by a modified Bligh and Dyer method and TLC analyses suggested that a hemolytic compound in the extract might be a type of lipid. These results suggest that a hemolytic lipid-like compound in an extract of H. marmoreus fruiting bodies may be released by a non-active precursor substance(s) through metalloenzyme(s) while the extract has a low pH.

Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

Assessment of the safety and applications of bacteriocinogenic Enterococcus faecium Y31 as an adjunct culture in North-eastern Chinese traditional fermentation paocai

Enterococcus faecium Y31 is a class IIa bacteriocin producer found in traditional Chinese fermented foods. The bacteriocin produced by Y31 (bacteriocin Y31) has a broad inhibitory spectrum. Here, we investigated the safety and behaviours of E. faecium Y31 in North-eastern Chinese traditional fermentation paocai (NCTFP) to assess its suitability as an adjunct culture. E. faecium Y31 showed interesting features, such as the absence of virulence determinants (aggregation substance [agg], enterococcal surface protein [esp], endocarditis antigen [efaAfm], gelatinase [gelE], hemolysin [cylA, cylB, cylM], sex pheromone [cpd, cob, ccf], adhesion of collagen [ace], and hyaluronidase [hyl] genes), sensitivity to various antibiotics, lack of haemolytic compounds, and lack of biogenic amine production. E. faecium Y31 could propagate in raw Chinese cabbage and produce bacteriocin Y31. To evaluate its performance as an adjunct culture for NCTFP, changes in the overall quality of control (noninoculated) and experimental NCTFP (inoculated with E. faecium Y31 under previously optimised fermentation conditions) were determined in duplicate fermentations of Chinese cabbage using chemical, microbiological, texture, and sensory evaluations. In the presence of E. faecium Y31, faster fermentation and higher bacteriocin Y31 production were observed than in the control. Additionally, the nitrite concentration of the fermentation solution decreased more rapidly, the total viability of the culture increased, and the lactic acid bacterium population was higher than those in NCTFP produced by spontaneous fermentation. No yeasts and moulds were detected in NCTFP produced by inoculation fermentation or spontaneous fermentation, and the textural properties of these two products were similar. Sensory evaluation results for NCTFP produced by inoculation fermentation were superior to those produced by spontaneous fermentation. Therefore, E. faecium Y31 was sufficient as an adjunct culture to enhance both the hygienic and sensory attributes of NCTFP and its use may help to address the issues inherent in commercial production of NCTFP.

Induced erythropoiesis during acute anemia in Atlantic salmon: A transcriptomic survey

Anemia is a common pathophysiological response to stressors, malnutrition and infections in salmonid fish. In order to improve our understanding of the molecular mechanisms and markers associated with induced erythropoiesis (EP) during acute anemia in Atlantic salmon (Salmo salar L.), we performed transcriptome analysis of fish injected with the hemolytic compound phenylhydrazine (PHZ). Treatment with a low dose of PHZ resulted in moderate but significant reduction of hematocrit (Hct) and increased transcription of cardiac erythropoietin (epo) at 2days post challenge (dpc), and epo receptor (epor) in spleen from 2 to 4dpc. Oligonucleotide microarrays were used to characterize the events of EP in the spleen, an important organ for expansive EP during acute erythropoietic stress in rodents, and these were compared to gene expression profiles of untreated mature red blood cells (RBC) in order to search for erythroid-specific genes. Splenic responses suggested a prevalence of protective mechanisms at the first stage, characterized by induced xenobiotic metabolism and responses to oxidative and protein stress. Erythroid-specific regulation was evident at 2dpc and enhanced by 4dpc, and gene expression profiles witnessed a rapid establishment of RBC phenotype although Hct levels remained low. A large group of genes showed a strong correlation to globins by expression profiles. In addition to epor this included genes of heme and iron metabolism, scavengers of free radicals and chaperones, channels and transporters, markers of erythrocytes, regulators of proliferation and cell cycle arrest and many genes with unidentified roles in RBC differentiation. Induced EP in spleen was characterized by specific features, such as upregulation of innate antiviral immune genes and sustained high expression of proapoptotic genes including caspases. Transcriptome changes suggested an association between EP and suppression of several developmental programs including adaptive immune responses. In conclusion, acute hemolysis and resulting anemia rapidly induced EP in the spleen of Atlantic salmon, which showed both common characteristics for all vertebrates as well as fish-specific properties.

Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7–40% from log phase growth to late log – early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20–31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60–90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC–MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity.

Toxicological studies of Karlodinium micrum (Dinophyceae) isolated from East China Sea

Karlodinium micrum (Strain NMBjah047) was isolated from the water samples of East China Sea (ECS). The hemolytic, ichthyotoxic, and cytotoxic activities of the algae was characterized. Embryotoxicity of both intra and extracellular extracts were also tested on a local sea urchin species. The algal intracellular hemolytic toxicity averaged about 87.5% at different algal growth phases. However, extracellular hemolytic activity depended on the population growth phase. The toxicity increased with the increase in the population size, reaching the highest hemolytic activity during the stationary phase, and maintained a relatively high activity even when the population declined. Time and density dependent ichthyotoxicity to Lateolabrax maculates juveniles was also detected. The LD50 in 24 h was 1.1 × 105 cells/mL. Inhibition of the fertilized egg hatching was also observed and estimated the IC50 in 40 h with 3.5 × 104 cells/mL. Extracellular extracts of K. micrum dense culture also showed significant cytotoxic activity on HUVEC (IC50 = 70.8 μg/mL). A dose dependent acute toxicity to embryos of sea urchin was also determined. The algal intracellular and extracellular extracts delayed or even restricted the embryological development of the sea urchin, illustrating the potential toxicity of K. micrum not only to vertebrates, but also to marine invertebrates. The hemolytic compounds in the ECS strain were extracted and analyzed. At least two fractions had significant hemolytic activities. A lipid-like compound, named Digalactosyldiacylglycerol (DGDG), was suggested to be responsible for the hemolytic activity in one of these fractions. From the results of the present studies, this strain of K. micrum isolated from the East China Sea might be considered a toxic strain with hemolytic activity, ichthyotoxicity, cytotoxicity and embryotoxicity.

Structure and Biosynthesis of Amphidinol 17, a Hemolytic Compound from Amphidinium carterae

Amphidinol 17 (AM17; 1), a novel amphidinol, has been isolated from a Bahamas strain of Amphidinium carterae. This new congener contains the signature hairpin region and a Delta(6) polyene arm, whereas the polyol arm is distinct from those of other amphidinols. The pattern of acetate incorporation in 1 was directly determined by feeding a single labeled substrate, [2-(13)C]acetate. While the highly conserved regions within the amphidinol family of AM17 have exhibited identical occurrences of cleaved acetates to other amphidinols for which the biosynthesis has been explored, the polyol arm for AM17 displays a higher degree of nascent chain processing that shows similarities to amphidinolide biosynthesis. AM17 exhibited an EC(50) of 4.9 microM in a hemolytic assay using human red blood cells but displayed no detectable antifungal activity.

Haemolytic activity within the species Fibrocapsa japonica (Raphidophyceae)

Strong haemolytic activity was observed for extracts of 15 Fibrocapsa japonica strains collected from different global regions. The EC 50 values ranged between 0.4 × 10 4 and 1.9 × 10 4 F. japonica cells ml −1. The relationship between the haemolytic activity observed in the cell extracts and the amount and nature of polyunsaturated fatty acids (PUFAs) was investigated. Between 7% and 89% of the haemolytic activity could be attributed to the amount and toxicity of the PUFAs C18:4n3 (OTA), C20:5n3 (EPA), and C20:4n6 (AA). However, the higher the haemolytic activity of a strain the more it appeared to be caused by haemolytic compounds other than the PUFA's. The EC 50 of the extracts with the highest haemolytic activity strongly depended on light intensity and was in line with observations made on Chattonella marina. In the cell extracts of the strains studied brevetoxins were always below detection limits. For strains where 20–35% of the haemolytic activity was not caused by PUFAs, the concentration of these compounds was close to LC 50 values reported for fish. Overall the haemolytic activity of the F. japonica strains was in the range of known harmful microalgae. Therefore, PUFAs in combination with potent light dependent haemolytic compounds could be a major cause of ichthyotoxicity observed during F. japonica blooms.

Anti-grazing properties of the toxic dinoflagellate Karlodinium veneficum during predator–prey interactions with the copepod Acartia tonsa

Karlodinium veneficum (syn. Karlodinium micrum, Bergholtz et al. 2006; J Phycol 42: 170-193) is a small athecate dinoflagellate commonly present in low levels in temperate, coastal waters. Occasionally, K. veneficum forms ichthyotoxic blooms due to the presence of cytotoxic, hemolytic compounds, putatively named karlotoxins. To evaluate the anti-grazing properties of these karlotoxins, we conducted food removal experiments using the cosmopolitan copepod grazer Acartia tonsa. Wild-caught, adult female A. tonsa were exposed to 6 monoalgal or mixed algal diets made using bloom concentrations of toxic (CCMP 2064) and non-toxic (CSIC1) strains of K. veneficum. Ingestion and clearance rates were calculated using the equations of Frost (1972). Exposure to the toxic strain of K. veneficum did not contribute to an increased mortality of the copepods and no sig- nificant differences in copepod mortality were found among the experimental diets. However, A. tonsa had significantly greater clearance and ingestion rates when exposed to a monoalgal diet of the non-toxic strain CSIC1 than when exposed to the monoalgal diet of toxic strain CCMP 2064 and mixed diets dominated by this toxic strain. These results support the hypothesis that karlotoxins in certain strains of K. veneficum deter grazing by potential predators and contribute to the formation and continuation of blooms.

Coelomic fluid proteins as basic components of innate immunity in earthworms

Coelomic fluid (CF) of earthworms contains cells and many molecular components involved in innate immunity. Glycoproteins of lectin character contribute to recognition of foreign material by binding to carbohydrates and cause its immobilisation and destruction by agglutination and lysis. Analysing several earthworm species ( Allolobophora chlorotica, Apporectodea caliginosa, Dendrodrillus rubidus, Eisenia fetida, Lumbricus rubellus, L. terrestris) we showed earlier that wounding and injection of foreign material into the coelomic cavity increases coelomocyte numbers and the activity of easily measurable humoral immune factors like agglutinins, lysins and proteases [I. Eue, E. Kauschke, W. Mohrig, E.L. Cooper, Isolation and characterization of earthworm hemolysins and agglutinins, Dev. Comp. Immunol. 22 (1998) 13--26; E. Kauschke, W. Mohrig, The occurrence of bacterioagglutinating, hemagglutinating and hemolytic compounds in the coelomic fluid of different European earthworm species, Zool. Jb. Physiol. 91 (1987) 467--477; E. Kauschke, P. Pagliara, L. Stabili, E.L. Cooper, Characterization of proteolytic activity in coelomic fluid of Lumbricus terrestris, L. Comp. Biochem. Physiol. 118B (2) (1997) 235--242; W. Mohrig, I. Eue, E. Kauschke, Proteolytic activities in the coelomic fluid of earthworms (Annelida, Lumbricidae), Zool. Jb. Physiol. 93 (1989) 303--317; W. Mohrig, I. Eue, E. Kauschke, F. Hennicke, Crossreactivity of hemolytic and heamagglutinating proteins in the coelomic fluid of earthworms, Comp. Biochem. Physiol. 115A (1) (1996) 19--30]. Earthworm CF proteases are relatively heat stable, posses an activity optimum at neutral to basic pH and are classified mainly as serine proteases [E. Kauschke, P. Pagliara, L. Stabili, E.L. Cooper, Characterization of proteolytic activity in coelomic fluid of Lumbricus terrestris, L. Comp. Biochem. Physiol. 118B (2) (1997) 235--242; W. Mohrig, I. Eue, E. Kauschke, Proteolytic activities in the coelomic fluid of earthworms (Annelida, Lumbricidae), Zool. Jb. Physiol. 93 (1989) 303--317]. Herein we focus on protease activity of the species A. caliginosa and L. rubellus, by characterizing activity parameters (time course, pH optimum, temperature sensitivity) and by comparing enzyme patterns of untreated and stimulated CF samples separated by PAGE. Disturbance of homeostasis in earthworms results in an increased immune response. Some of the immune proteins might be considered as suitable biomarkers in monitoring environmental conditions. Especially protease pattern and protease activity may be considered as promising candidates in that regard and are easy to analyse.

A Toxicogenomic Approach Revealed Hepatic Gene Expression Changes Mechanistically Linked to Drug-Induced Hemolytic Anemia

A variety of pharmaceutical compounds causes hemolytic anemia as a significant adverse effect and this toxicity restricts the clinical utility of these drugs. In this study, we applied microarray technology to investigate hepatic gene expression changes associated with drug-induced hemolytic anemia and to identify potential biomarker genes for this hematotoxicity. We treated female Sprague-Dawley rats with two hemolytic anemia-inducing compounds: phenylhydrazine and phenacetin. Hepatic gene expression profiles were obtained using a whole-genome oligonucleotide microarray with pooled RNA samples from individual rats within each dose group and analyzed in comparison with hepatic histopathology, hematology, and blood chemistry data. We identified a small subset of genes that were commonly deregulated in all the severe hemolytic conditions, some of which were considered to be involved in hepatic events characteristic of hemolytic anemia, such as hemoglobin biosynthesis, heme metabolism, and phagocytosis. Among them, we selected six upregulated genes as putative biomarkers, and their expression changes from microarray measurements were confirmed by quantitative real-time PCR using RNAs from individual animals. They were Alas2, beta-glo, Eraf, Hmox1, Lgals3, and Rhced. Expression patterns of all these genes showed high negative and positive correlation against erythrocyte counts and total bilirubin levels in circulation, respectively, suggesting that these genes may be the potential biomarkers for hemolytic anemia. These findings indicate that drug-induced hemolytic anemia may be detected based on hepatic changes in the expression of a subset of genes that are mechanistically linked to the hematotoxicity.

A modified assay to determine hemolytic toxin variability among Karenia clones isolated from the Gulf of Mexico

Karenia brevis (formerly Gymnodinium breve) is a toxic marine dinoflagellate generally restricted to the Gulf of Mexico and is the main causative organism in fish kills, shellfish intoxications and respiratory distress in humans following bloom events. K. mikimotoi is a morphologically similar co-occurring species which is toxic in other parts of the world oceans, but has not been recognized as a major contributor in toxicity of blooms within the Gulf of Mexico. Recently there has been increasing evidence of the simultaneous production of a variety of bioactive compounds in addition to potent neurotoxins (brevetoxin) in Karenia brevis isolates. These compounds are potentially ichthyotoxic and have been shown to cause hemolysis in several bioassays [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519; Kirkpatrick, B., Fleming, L.E., Squicciarini, D., Backer, L.C., Clark, R., Abraham, W., Benson, J., Cheng, Y.S., Johnson, D., Pierce, R., Zaias, J., Bossart, G.D., Baden, D.G., 2004. Literature review of Florida red tide: implications for human health effects. Harmful Algae 3, 99–115]. Presence of hemolytic compounds may therefore add to the overall toxicity levels of bloom events. Current monitoring methods include assays which are highly sensitive in brevetoxin detection and yet may not target other harmful compounds. By adapting protocols developed by Eshbach et al. [Eshbach, E., Scharsack, J., John, U., Medlin, L., 2001. Improved erythrocyte lysis assay in microtitre plates for the sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol. 21, 513–519], Red drum ( Sciaenops ocellatus) erythrocytes were used to create a modified bioassay to detect hemolytic activity of crude algal extracts. Red drum was selected because it is endemic to coastal areas throughout the Gulf of Mexico and is sensitive to Karenia blooms, and thus makes this species a valid ecological target. Preliminary data has shown this method is sensitive for use in assessing hemolysis induced by laboratory cultures down to levels of 1 × 10 3 cells mL −1. Results showed an unexpectedly high level of hemolytic activity among K. mikimotoi clones, with one Texas strain inducing significantly higher hemolysis compared to Florida K. brevis isolates. Using this approach, future research efforts will examine the difference in production of hemolytic compounds among various Karenia clones.

Purification and characterization of photosensitizing hemolytic toxin from harmful red tide phytoplankton, Heterocapsa circularisquama

We have previously found that the methanol extract prepared from Heterocapsa circularisquama, a harmful red rid dinoflagellate, showed light-dependent hemolytic activity toward rabbit erythrocytes. Interestingly, the cytotoxicity of the extract against HeLa cells was also strictly light-dependent, and no significant toxic effect was observed even at very high concentration in the dark. In this study, the hemolytic agents present in the extract were purified by several chromatographic procedures. Final HPLC analysis revealed that there are at least two hemolytic toxins with different retention times (H2-a and H3-a), which have similar absorption spectra. Although H3-a fraction was contaminated by a trace amount of H2-a, H2-a fraction gave a single peak on a three-dimensional chromatogram. The results of fast atom bombardment (FAB)–mass spectrometry (MS) and electrospray ionization quadrupole time-of-flight (ESI Q-TOF) mass spectrometry analyses suggested that the molecular weight of H2-a was 566. Since nuclear magnetic resonance analysis indicated the presence of a pyrrole ring in H2-a molecule, H2-a may be a porphyrin derivative with similar structure of pyropheophorbide a methyl ester, a well-known photosensitizing hemolytic compound. However, some structural and physicochemical differences were observed between H2-a and pyropheophorbide a methyl ester. Since H2-a showed potent photosensitizing cytotoxicity toward HeLa cells in a concentration-dependent manner as well as hemolytic activity, H2-a may be a novel porphyrin derivative with photosensitizing hemolytic cytotoxicity.

The structures of five new antifungal and hemolytic amphidinol analogs from Amphidinium carterae collected in New Zealand

Amphidinols, antifungal and hemolytic compounds, were isolated from the marine dinoflagellates Amphidinium spp. Their structures were characterized by a conjugated triene, two ether rings, and polyhydroxy groups. Five new amphidinol analogs, amphidinols 9, 10, 11, 12 and 13 were isolated together with amphidinol 2 (AM2), and amphidinol 4 (AM4) from the dinoflagellate, Amphidinium carterae collected in New Zealand. Their structures were elucidated by detailed analyses of MS and 2D NMR spectra. Amphidinol 9 was a regioisomer of AM3, and amphidinol 10 was dinorAM4. Amphidinol 11, 12 and 13 possess a sulfate ester at C1 of AM2, AM4, and AM9, respectively. Attachment of the sulfate ester markedly reduced their antifungal and hemolytic activities.

Low-molecular-weight, biologically active compounds from marine Pseudoalteromonas species.

We have examined the ability of marine Proteobacteria from the Pseudoalteromonas genus and Alteromonas macleodii to produce low-molecular-weight, biologically active compounds with antimicrobial and surface-active properties. A new marine bacterium, Pseudoalteromonas issachenkonii, exhibited a high level of biological activity and produced antifungal and hemolytic compounds. A detailed spectroscopic investigation based on UV, IR, high-resolution mass spectrometry, and 2D 1H and 13C nuclear magnetic resonance revealed that the former was indole-2,3-dione (isatin). The chemical structure of red-brown pigment (C9H7N3OS3) responsible for hemolytic activity remained unclear. Four of the 15 strains studied (P. luteoviolacea, P. rubra, P. undina, and P. issachenkonii) produced cell-bound, two (P. elaykovii and P. carrageenovora) produced extracellular, and one strain (P. citrea) produced cell-bound and extracellular fatty acids and phospholipids with surface activity. Neither peptides nor glycolipids with surface activity were detected.