Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Identification of fatty acid amides in human plasma

A family of five long-chain fatty acid carboxamides has been identified and semi-quantified in human plasma by GC-MS. One saturated and four unsaturated amides were found. Luteal phase plasma from 16 women was studied, and all five of the amides were found in ten of the subjects, but none in the other six. The structure of these endogenous amides was established by comparing their GC and MS characteristics with those of the synthetic amides prepared by ammonolysis of corresponding long-chain fatty acid acyl chlorides.     

AFLP fingerprinting shows that a single Prymnesium parvum harmful algal bloom consists of multiple clones

Due to slow rates of molecular evolution, DNA sequences used to identify and build phylogenies of algal species involved in harmful algal blooms (HABs) are generally invariant at the intraspecific level. This means that it is unknown whether HAB events result from the growth of a single clone, a few dominant clones, or multiple clones. This is true despite the fact that several physiological and demographic traits, as well as toxicity, are known to vary across clones. We generated AFLP fingerprints from a set of 6 clonal isolates, taken from a bloom of Prymnesium parvum at a striped bass mariculture facility. This new haptophyte bloom was recently implicated in fish kills at several sites in the United States. The AFLP fragments were highly reproducible and showed that all isolates were distinguishable due to abundant AFLPs unique to single isolates. These results demonstrate that blooms can be genetically diverse outbreaks and indicate that AFLP can be a powerful molecular tool for characterizing and monitoring this diversity.     

Fatty acid amides from freshwater green alga Rhizoclonium hieroglyphicum

Freshwater green algae Rhizoclonium hieroglyphicum growing in the Ural Mountains were examined for their fatty acid amides using capillary gas chromatography-mass spectrometry (GC-MS). Eight fatty acid amides were identified by GC-MS. (Z)-9-octadecenamide was found to be the major component (2.26%).     

Absolute configuration at C14 and C85 in prymnesin-2, a potent hemolytic and ichthyotoxic glycoside isolated from the red tide alga Prymnesium parvum

Prymnesin-2 is a potent red tide toxin characterized by a highly oxidized C(90) carbon chain and multiple functional groups. Succeeding the assignment of the relative stereochemistry in the polycyclic-ether segment, the absolute configurations of two chiral centers in linear chain parts were elucidated. The configuration at C14 bearing an amino group was determined to be S by using a chiral anisotropic reagent and that at chlorinated C85 to be S by fluorimetric chiral HPLC comparison between a degradation product and synthetic references.     

Photoinactivation of Ichthyrotoxin from Axenic Cultures of Prymnesium parvum Carter

The ichthyotoxin of Prymnesium parvum is inactivated by visible (400 to 510 mμ) as well as by ultraviolet light (255 mμ). The changes in the absorption spectrum of purified (pigment-free) ichthyotoxin during this process indicate that different mechanisms may be involved in the inactivation by visible or ultraviolet light. Photoinactivation is not affected by the presence of cells, cell pigments, oxygen, or glutathione.     

Acyl-CoA: cholesterol acyltransferase inhibitory activities of fatty acid amides isolated from Mylabris phalerate Pallas

Unsaturated fatty acid amides, 9(Z)-octadecenamide (2) and 9(Z),12(Z)-octadecadienamide (4) as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT) were isolated from the ethyl acetate extracts of the insect, Mylabris phalerate Pallas, and elucidated by their spectroscopic data analysis. Compounds 2 and 4 inhibited rat liver microsomal ACAT, hACAT-1, and hACAT-2 with IC(50) values of 170, 85, and 63 microM for 2 and of 151, 53, and 45 microM for 4, respectively.     

Occurrence of toxic Prymnesium parvum blooms with high protease activity is related to fish mortality in Hungarian ponds

The unicellular alga Prymnesium parvum has been responsible for toxic incidents with severe ecological impacts in many parts of the world, and causes massive fish kills worldwide. Recently the haptophyte microalgae have caused water-bloom (4.3 × 10⁴ cells ml⁻¹) in 6 fish ponds with high conductivity in Hungary, and caused fish mortality with typical symptoms. Toxicity of P. parvum from water samples was quantified by the assay of the influence of its cell-free filtrates on haemolysis (346 ± 42.2) and in fish and daphnia toxicity tests. High amount of proteases in P. parvum containing waterbloom samples were detected with the help of activity gel electrophoresis. The proteases of investigated P. parvum samples (125–18 kDa) showed high gelatinolytic activity and some of them showed sensitivity to EDTA (inhibitors of metalloproteases) and to PMSF (inhibitors of serine proteases).     

Retrospective analysis of associations between water quality and toxic blooms of golden alga (Prymnesium parvum) in Texas reservoirs: Implications for understanding dispersal mechanisms and impacts of climate change

Toxic blooms of golden alga (GA, Prymnesium parvum) in Texas typically occur in winter or early spring. In North America, they were first reported in Texas in the 1980s, and a marked range expansion occurred in 2001. Although there is concern about the influence of climate change on the future distribution of GA, factors responsible for past dispersals remain uncertain. To better understand the factors that influence toxic bloom dispersal in reservoirs, this study characterized reservoir water quality associated with toxic GA blooms since 2001, and examined trends in water quality during a 20-year period bracketing the 2001 expansion. Archived data were analyzed for six impacted and six nonimpacted reservoirs from two major Texas basins: Brazos River and Colorado River. Data were simplified for analysis by pooling spatially (across sampling stations) and temporally (winter, December–February) within reservoirs and generating depth-corrected (1 m) monthly values. Classification tree analysis [period of record (POR), 2001–2010] using salinity-associated variables (specific conductance, chloride, sulfate), dissolved oxygen (DO), pH, temperature, total hardness, potassium, nitrate+nitrite, and total phosphorus indicated that salinity best predicts the toxic bloom occurrence. Minimum estimated salinities for toxic bloom formation were 0.59 and 1.02 psu in Brazos and Colorado River reservoirs, respectively. Principal component analysis (POR, 2001–2010) indicated that GA habitat is best defined by higher salinity relative to nonimpacted reservoirs, with winter DO and pH also being slightly higher and winter temperature slightly lower in impacted reservoirs. Trend analysis, however, did not reveal monotonic changes in winter water quality of GA-impacted reservoirs during the 20-year period (1991–2010) bracketing the 2001 dispersal. Therefore, whereas minimum levels of salinity are required for GA establishment and toxic blooms in Texas reservoirs, the lack of trends in water quality suggests that conditions favorable for toxic blooms pre-date the 2001 expansion. These observations are consistent with a climate change-independent scenario of past GA dispersals in Texas reservoirs driven by novel introductions into pre-existing favorable habitat. Reports of latent GA populations in certain nonimpacted reservoirs, however, provide a plausible scenario of future dispersals characterized by prolonged periods between colonization and toxic bloom development and driven by changes in water quality, natural, or anthropogenic.     

Current progress in the fatty acid metabolism in Cryptosporidium parvum

Cryptosporidium parvum is one of the apicomplexans that can cause severe diarrhea in humans and animals. The slow development of anti-cryptosporidiosis chemotherapy is primarily due to the poor understanding on the basic metabolic pathways in this parasite. Many well-defined or promising drug targets found in other apicomplexans are either absent or highly divergent in C. parvum. The recently discovered apicoplast and its associated Type II fatty acid synthetic enzymes in Plasmodium, Toxoplasma, and Eimeria apicomplexans are absent in C. parvum, suggesting this parasite is unable to synthesize fatty acids de novo. However, C. parvum possesses a giant Type I fatty acid synthase (CpFAS1) that makes very long chain fatty acids using mediate or long chain fatty acids as precursors. Cryptosporidium also contains a Type I polyketide synthase (CpPKS1) that is probably involved in the production of unknown polyketide(s) from a fatty acid precursor. In addition to CpFAS1 and CpPKS1, a number of other enzymes involved in fatty acid metabolism have also been identified. These include a long chain fatty acyl elongase (LCE), a cytosolic acetyl-CoA carboxylase (ACCase), three acyl-CoA synthases (ACS), and an unusual "long-type" acyl-CoA binding protein (ACBP), which allows us to hypothetically reconstruct the highly streamlined fatty acid metabolism in this parasite. However, C. parvum lacks enzymes for the oxidation of fatty acids, indicating that fatty acids are not an energy source for this parasite. Since fatty acids are essential components of all biomembranes, molecular and functional studies on these critical enzymes would not only deepen our understanding on the basic metabolism in the parasites, but also point new directions for the drug discovery against C. parvum and other apicomplexan-based diseases.     

Characterization of cross-linked hydroxycinnamic acid amides isolated from potato common scab lesions

Four feruloyl amides, N-trans-feruloyloctopamine (1), N-cis-feruloyloctopamine (2), N-trans-feruloyltyramine (3), N-cis-feruloyltyramine (4), a cross-linked N-trans-feruloyltyramine dimer (5), and a cross-linked N-cis-feruloyltyramine dimer (6) were isolated from potato common scab lesions. The compounds were purified by TLC and characterized by a combination of (1)H and (13)C NMR spectroscopic techniques. The presence of an accompanying minor complex of cross-linked dimers containing both feruloyltyramines and feruloyloctopamines was also demonstrated. This is the first characterization of cross-linked hydroxycinnamic acid amides associated with wound healing in potato (Solanum tuberosum) tubers.     

Growth and Colony Formation of the Phytoflagellate Prymnesium parvum Carter on Solid Media

SYNOPSIS. Colonies of Prymnesium parvum from single cells were obtained in an atmosphere of high humidity, in soft-agar overlayers on solid medium. Light intensity above 60 foot candles at the surface of the agar plates inhibited colonial development. Such colonies were used to obtain pure lines of the phytoflagellate.
A method for direct demonstration of hemolysin formation on agar plates was developed. The occurrence of hemolytic activity by P. parvum colonies was light-dependent.     

Synthesis and antituberculosis activity of new fatty acid amides

This work reports the synthesis of new fatty acid amides from C16:0, 18:0, 18:1, 18:1 (OH), and 18:2 fatty acids families with cyclic and acyclic amines and demonstrate for the first time the activity of these compounds as antituberculosis agents against Mycobacterium tuberculosis H₃₇Rv, M. tuberculosis rifampicin resistance (ATCC 35338), and M. tuberculosis isoniazid resistance (ATCC 35822). The fatty acid amides derivate from ricinoleic acid were the most potent one among a series of tested compounds, with a MIC 6.25 μg/mL for resistance strains.     

Potentially toxic and harmful phytoflagellates from the Mexican Pacific coasts

The phytoflagellates are a heterogeneous group of autotrophic, heterotrophic and mixothrophic flagellates of trophic importance in several ecosystems. As in the rest of Latin America, the phytoflagellates that occur in the Mexican Pacific coasts are virtually unknown except for a few records. Their study require complicated collection and analysis methods, a probable cause for the scarce knowledge of this group in tropical and subtropical areas. Material recently collected from various localities along the Mexican Pacific coasts was used to study phytoflagellates, including toxic and potentially toxic species. Plankton samples were treated by gravity and pump filtration, using different methods for fixation and analysis. The phyla Euglenophyta, Heterokontophyta and Haptophyta were found. They occur as plankton in oceanic and shallow coastal waters.