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Biomedical subjects

W W Carmichael

Publications and source records attributed to W W Carmichael.

At least 37 records · Page 2Linked to original sources
PubMed1994

Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins.

Microcystins and nodularins are cyclic peptide hepatotoxins and tumor promoters produced by several genera of cyanobacteria. Using a rabbit anti-microcystin-LR polyclonal antibody preparation, the cross-reactivity with 18 microcystin and nodularin variants was tested. A hydrophobic amino acid, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E),6(E)-dienoic acid (Adda), which has the (E) form at the C-6 double bond in both microcystin and nodularin, was found essential for these toxins to express antibody specificity. Modification of -COOH in glutamic acid of microcystin and nodularin did not alter their antigenicity. Antibody cross-reactivity of these toxins was compared with their ability to inhibit protein phosphatase type 1 (PP1). Detection of PP1 inhibition was done by measuring the inhibition effect of the toxins on p-nitrophenol phosphate activity toward PP1. PP1 was obtained as recombinant PP1 expressed in E. coli. The inhibition effect of five microcystins and two nodularins on recombinant PP1 activity toward p-nitrophenol phospate was measured in a microwell plate reader. The concentration of microcystin-LR causing 50% inhibition of recombinant PP1 activity (IC50) was about 0.3 nM, while that of two modified microcystins had a significantly higher IC50. Microcystin-LR and nodularin with the (z) form of Adda at the C-6 double bond or having the monoester of glutamic acid did not inhibit PP1. These three toxins were also nontoxic in the mouse bioassay. These results show the importance of Adda and glutamic acid in toxicity of these cyclic peptides and that PP1 inhibition is related to the toxins' mechanism of action.

Amino Acids↗
PubMed1993

Structural modifications imparting reduced toxicity in microcystins from Microcystis spp.

A cyanobacterial (blue-green algal) bloom containing Microcystis aeruginosa (dominant), M. viridis, and M. wesenbergii, was collected from Homer Lake (Illinois, U.S.A.) in the summer of 1988 and microcystins were isolated. One microcystin of substantially reduced toxicity was isolated, together with ten hepatotoxic microcystins. The compound with reduced toxicity was nonlethal at 1 mg/kg (i.p. mouse) and was determined to have a (C3H7O2) mono-ester of the alpha-carboxyl on the Glu unit of microcystin-LR. The other nine microcystins apart from MCLR had approximate LD50S ranging from 97 micrograms/kg to 750 micrograms/kg.

Amino Acid Sequence↗
PubMed1992

Liver tumor promotion by the cyanobacterial cyclic peptide toxin microcystin-LR.

Certain waterblooms of toxic cyanobacteria (blue-green algae) are a health threat because of their production of toxic peptides, termed microcystins, which cause liver damage in wild and domesticated animals. The most widely studied microcystin is microcystin-LR, a heptapeptide containing the two L-amino acids, leucine and arginine. The inhibition of protein phosphatase type 1 and type 2A activities by microcystin-LR is similar to that of the known protein phosphatase inhibitor and tumor promoter okadaic acid. We show in this report that microcystin-LR, applied below the acute toxicity level, dose-dependently increases the number and percentage area of positive foci for the placental form of glutathione S-transferase in rat liver, which was initiated with diethylnitrosamine. The result was obtained independently through two animal experiments. This observation indicates that microcystin-LR is a new liver tumor promoter mediated through inhibition of protein phosphatase type 1 and type 2A activities. This provides further evidence that the okadaic acid pathway is a general mechanism of tumor promotion in various organs, such as mouse skin, rat glandular stomach and rat liver.

Alanine Transaminase↗
PubMed1992

Structure determination and toxicity of a new microcystin from Microcystis aeruginosa strain 205.

A new hepatotoxic microcystin was isolated from the cyanobacterium Microcystis aeruginosa strain 205. Its structure was found to be [Dha7]microcystin-RR as determined by amino acid analysis, mass spectrometry and 1H NMR spectroscopy. LD50 value (i.p. mouse) of this toxin was 180 micrograms/kg. The 48 hr lethal concentration (48-hr-LC50) of the toxin for larvae of the yellow fever mosquito, Aedes aegypti, was 14.9 micrograms/ml.

Aedes↗
PubMed1992

Two new L-serine variants of microcystins-LR and -RR from Anabaena sp. strains 202 A1 and 202 A2.

Two new microcystins, [L-Ser7]microcystin-LR (1) and [L-Ser7]microcystin-RR (2), were isolated from a filamentous fresh water cyanobacterium (blue-green alga), Anabaena sp. strain 202 A1, along with the two major toxins, [Dha7]microcystin-LR (3) and [Dha7]microcystin-RR (4) and their minor components the D-Asp variants [D-Asp3,Dha7]microcystin-LR (5) and [D-Asp3,Dha7]microcystin-RR (6). Anabaena sp. strain 202 A1 also produced another new toxin, whose structure is tentatively proposed as [D-Asp3,L-Ser7]microcystin-XR (7), where X is a leucine homologue. Anabaena sp. strain 202 A2 produced one new microcystin, 1, and three known microcystins, 3, 4, and 5. The structures of the toxins were assigned based on their amino acid analyses, and fast atom bombardment mass spectrometry data.

Amino Acid Sequence↗
PubMed1992

Two methyl ester derivatives of microcystins, cyclic heptapeptide hepatotoxins, isolated from Anabaena flos-aquae strain CYA 83/1.

Cultured cells of Anabaena flos-aquae strain CYA 83/1, isolated from Lake Edlandsvatn, Norway, produced two microcystin mono-methyl ester derivatives (1 and 2) at the D-Glu unit in addition to microcystin-LR (3), [D-Asp3]microcystin-LR (4), microcystin-RR (5), and [D-Asp3]microcystin-RR (6). Structures of these compounds were assigned based on their amino acid analysis with a Waters Pico Tag HPLC system plus fast atom bombardment mass spectrometry (FABMS), including tandem FABMS, analysis on the two new microcystins, [D-Glu(OCH3)6]microcystin-LR (1) and [D-Asp3, D-Glu(OCH3)6]microcystin-LR (2). Toxicity data were not obtained for 1 and 2 because of the small amounts isolated from the cells.

Amino Acid Sequence↗
PubMed1992

Isolation and structures of microcystins from a cyanobacterial water bloom (Finland).

A hepatotoxic cyanobacterial (blue-green algal) water bloom was collected from a constructed water reservoir in Finland. The water bloom contained two cyanobacterial species, Microcystis aeruginosa and Aphanizomenon flos-aquae. Two hepatotoxins, 1 and 2, were isolated from extracts of lyophilized cells. The structures of 1 and 2 were assigned based upon their amino acid analyses on a Waters Pico Tag HPLC system and a chiral GC capillary column (Chirasil Val III), fast atom bombardment mass spectrometry (FABMS), high resolution FABMS, and tandem FABMS data. Toxin 1 was identical to a previously reported compound, [D-Asp3]microcystin-RR. Toxin 2 was new and was assigned the structure [D-Asp3]microcystin-YR.

Amino Acids↗
PubMed1992

Isolation and structures of five microcystins from a Russian Microcystis aeruginosa strain CALU 972.

Five microcystins were obtained from Microcystis aeruginosa strain CALU 972 isolated from a hepatotoxic water bloom collected in Lake Kroshnosero (Russia). The structure of a new toxin (1) was determined as [Dha7]microcystin-YR by amino acid analyses and fast atom bombardment mass spectrometry, and the toxins 2, 3, 4, and 5 were assigned the structures as [Dha7]microcystin-LR, [D-Asp3,Dha7]microcystin-LR, [Dha7]microcystin-RR, and [D-Asp3,Dha7]microcystin-RR, respectively, by direct comparison with authentic samples.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena.

Hepatotoxins (microcystins) from seven freshwater Anabaena strains originating from three different Finnish lakes and one lake in Norway were isolated by high-performance liquid chromatography and characterized by amino acid analysis and fast atom bombardment mass spectrometry. All strains produced three to seven different microcystins. A total of 17 different compounds were isolated, of which 8 were known microcystins. The known compounds identified from six strains were MCYST (microcystin)-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3,Dha7]MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. With the exception of MCYST-LR and [D-Asp3]MCYST-LR, this is the first time that isolation of these toxins from Anabaena strains has been reported. Three of the strains produced one to three toxins as minor components which could not be identified. Anabaena sp. strain 66 produced four unidentified toxins. The other Anabaena strains always contained both MCYST-LR and MCYST-RR and/or their demethyl variants. Quantitative differences between toxins within and between strains were detected; at times MCYST-LR and at other times MCYST-RR or demethyl derivatives thereof were the most abundant toxins found in a strain.

Amino Acid Sequence↗
PubMed1991

Anatoxin-a(s), a naturally occurring organophosphate, is an irreversible active site-directed inhibitor of acetylcholinesterase (EC 3.1.1.7).

Anatoxin-a(s) is a guanidine methyl phosphate ester (unprotonated molecular ion equals 252 daltons) isolated from the freshwater cyanobacterium (blue-green alga) Anabaena flos-aquae strain NRC 525-17. Previous work has shown anatoxin-a(s) to be a potent irreversible inhibitor of electric eel acetylcholinesterase (EC 3.1.1.7, AChE). In the present study the interaction of anatoxin-a(s) with AChE was investigated by protection studies and since similarities have been noted between anatoxin-a(s) and the synthetic organophosphate anticholinesterases, the ability of reactivators to reactivate the inhibited enzyme was investigated. Treatments directed toward eliminating poisoning symptoms and in vivo protection from anatoxin-a(s) poisonings were investigated using oxime reactivators and atropine or pretreatment with a carbamate and atropine. Anatoxin-a(s) was shown to be an active site-directed inhibitor of acetylcholinesterase which is resistant to oxime reactivation due to the structure of its enzyme adduct. In vivo pretreatment with physostigmine and high concentrations of 2-PAM were the only effective antagonists against a lethal dose of anatoxin-a(s).

Acetylcholinesterase↗
PubMed1991

Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver.

Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol. wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to [3H]-dihydro-microcystin-LR ( [3H]-2HMC-LR), and purified to greater than 99% purity by C-18 reverse-phase high-performance liquid chromatography. The uptake and subcellular distribution of [3H]-2HMC-LR were determined in suspensions of hepatocytes at 0 degrees C and 37 degrees C, or following rifampicin pretreatment, and in perfused rat liver. The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation. Suspensions of 7.5 x 10(6) hepatocytes also were incubated with 10 micrograms/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin). Isolated rat livers were perfused with media containing [3H]-2HMC-LR and the uptake of radiolabel was determined. Sequential biopsy samples were collected for histologic examination. The remaining liver was homogenized and subcellular fractions prepared. Uptake of radiolabel was rapid in both cell suspension at 37 degrees C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0 degrees C and by rifampicin (50 micrograms/ml) pretreatment. Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with [3H]-2HMC-LR. In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction. In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions. Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein. Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein. These data suggest that the uptake of [3H]-2HMC-LR occurs primarily by an energy-dependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).

Animals↗
PubMed1991

Morphologic and staining characteristics of a cyanobacterium-like organism associated with diarrhea.

A spherical organism 9-10 microns in diameter, seen in three outbreaks of diarrhea in Southeast Asia and the United States during the past 2 years, bore characteristics of a cyanobacterium when observed in formalin-preserved stool specimens and by electron microscopy. Organisms in freshly passed stool specimens showed an internal morula of lipid-containing globules. In fresh water, the morula divided into two sausage-shaped structures resembling the sporocysts of an isosporid coccidian. After 7 months, the organisms had not developed the crescentic sporozoites seen in the Coccidia but had begun to multiply slowly in culture. It was impossible to stain the internal structures of the organisms because the outer cyst wall ruptured during desiccation, releasing the contents of the cysts. The organisms were readily identified by their intense blue autofluorescence under UV light, but they were also recognizable by bright-field microscopy and by a modified acid-fast stain. Almost all infected persons suffered intermittent diarrhea for 2-3 weeks and many emphasized a feeling of intense fatigue during the course of their illness.

Asia, Southeastern↗
PubMed1991

Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa.

The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.

Actins↗
PubMed1990

In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and fibroblasts.

Three microcystins, YR, LR and RR and nodularin, all of which are hepatotoxic compounds, inhibited dose-dependently the activity of protein phosphatase 2A in and the specific [3H]okadaic acid binding to a cytosolic fraction of mouse skin, as strongly as okadaic acid. However, microcytins and nodularin did not induce any effects on mouse skin or primary human fibroblasts. Microinjection of microcystin YR into primary human fibroblasts induced morphological changes which were induced by incubation with okadaic acid. Microcystins and nodularin penetrate into the epithelial cells of mouse skin and human fibroblasts with difficulty, which reflects tissue specificity of the compounds.

Animals↗
PubMed1990

Inhibition of protein phosphatases by microcystins and nodularin associated with hepatotoxicity.

Microcystins and nodularin, isolated from toxic blue-green algae, are hepatotoxic monocyclic polypeptides. Both microcystins and nodularin inhibited in vitro protein phosphatase activity present in a cytosolic fraction of mouse liver, bound to the okadaic acid receptors, protein phosphatases 1 and 2A, and thus resulted in the increase of phosphoproteins; this was referred to as the apparent "activation" of protein kinases. Their concentrations causing 50% of the maximal effects are comparable to that of okadaic acid, a potent protein phosphatase inhibitor and a potent tumor promoter, in the nanomolar range of concentration. The increase of phosphoproteins was observed in rat primary cultured hepatocytes and was subsequently associated with morphological changes, which appeared to be a step in the process of hepatotoxicity. The well-known hepatotoxic compounds, alpha-amanitin and phalloidin, did not show any effects similar to those of microcystins, nodularin and okadaic acid. It is suggested that the hepatotoxicity of microcystins and nodularin may result from inhibition of protein phosphatases and the increase of phosphoproteins.

Animals↗