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Biomedical subjects

A M Dahlem

Publications and source records attributed to A M Dahlem.

At least 19 recordsLinked to original sources
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↗
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

Reversal of cholinesterase inhibition and clinical signs and the postmortem findings in mice after intraperitoneal administration of anatoxin-a(s), paraoxon or pyridostigmine.

The reversibility of inhibition of plasma, red blood cell (RBC), and diaphragm cholinesterase (ChE) and clinical signs in mice given anatoxin-a(s) [antx-a(s)], a ChE inhibitor from Anabaena flos-aquae NRC-525-17, were characterized and compared with the effects of 2 known ChE inhibitors, the organophosphorus compound paraoxon and the carbamate pyridostigmine bromide. To follow recovery of ChE activity, mice were given either a control solution or an LD40 dose of one of the toxicants ip and killed at time points up to 8 d postdosing. After dosing, mice were monitored for diarrhea, fasciculations, respiratory difficulty, salivation, and tremors. In general, clinical signs in mice given antx-a(s) persisted longer than in mice given pyridostigmine and were more similar in duration to the clinical signs in mice given paraoxon. Histologic lesions were not detected in tissues of mice killed after administration of antx-a(s). Anatoxin-a(s) inhibited lesions were diaphragm ChE for greater than 1 but less than 2 d and RBC ChE for 8 d. The time required for recovery from Antx-a(s)-induced inhibition of ChE in plasma, RBC, and diaphragm was similar to or longer than that with paraoxon and longer than that with pyridostigmine. Based on the duration of antx-a(s) induced clinical signs and ChE inhibition in mice, antx-a(s) appears to be an in vivo irreversible inhibitor of ChE.

Animals↗
PubMed1990

Isolation and characterization of the minor components associated with microcystins LR and RR in the cyanobacterium (blue-green algae).

Two structurally similar analogues of microcystins LR and RR, cyclic peptide hepatotoxins from Microcystis, were isolated by chromatographic methods. Although they have the same mol. wt and amino acid compositions as those of the parent toxins, they do not possess similar toxicities. Ultraviolet and 1H-NMR spectral data for both components demonstrate clear structural difference of these cyclic peptides from the parent toxins, which are probably responsible for the marked decreases in their observed toxicities.

Amino Acids↗
PubMed1990

Synthesis, organotropism and hepatocellular uptake of two tritium-labeled epimers of dihydromicrocystin-LR, a cyanobacterial peptide toxin analog.

Two tritium-labeled epimers of dihydromicrocystin-LR, a derivative of the cyanobacterial peptide hepatotoxin microcystin-LR, were synthesized by reduction with sodium boro[3H]hydride and purified with reversed-phase liquid chromatography. The epimers were hepatotoxic in mice; the i.p. LD50 was 120-135 micrograms/kg. They were concentrated in the liver and to some extent in the intestine and the kidney after an i.v. injection. Freshly isolated rat hepatocytes showed a rapid uptake of both epimers. The cellular uptake of the epimers was almost complete within 5 min at concentrations 1 microM (0.5 microM dihydromicrocystin-LR + 0.5 microM microcystin-LR) and 4 microM (0.5 microM + 3.5 microM). The uptake of the earlier eluting epimer was about three times higher than that of the later eluting epimer.

Animals↗
PubMed1990

Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152.

A strain of the filamentous cyanobacterium Nostoc sp. isolated from a lake in Finland was found to produce at least nine hepatotoxic peptides with chemical and toxicological properties similar to those of the hepatotoxic hepta- and pentapeptides produced by other cyanobacteria. Toxins were isolated and purified by high-performance liquid chromatography. Amounts available for five of the purified toxins (P6, P14, P15, P16, and P18) were adequate for high-performance liquid chromatography amino acid analysis and determination of molecular weight by fast-atom bombardment-mass spectrometry (FAB-MS). Quantities of three toxins (P14, P15, and P16) were adequate for further analysis by high-resolution FAB-MS, FAB-MS/MS, and 1H-nuclear magnetic resonance. Analysis showed that the toxins are new types of microcystin-LR homologs. Microcystin-LR contains equimolar amounts of D-alanine, L-leucine, D-erythro-beta-methylaspartic acid, L-arginine, ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), D-glutamic acid, and N-methyldehydroalanine (molecular weight [MW], 994). Nostoc sp. strain 152 was found to produce the following microcystin-LR homologs: (i) P6 contains an extra methylene group most probably due to the presence of N-methyldehydrobutyrine instead of N-methyldehydroalanine (MW, 1,008); (ii) P14 is O-acetyl-O-demethyl ADDA-microcystin-LR (MW, 1,022); (iii) P15 is 3-demethyl-O-acetylADDA-homoarginine-microcystin-LR (MW, 1,036); (iv) P16 is 3-demethyl-O-acetyl-O-demethylADDA-microcystin-LR (MW, 1,008); (v) P18 is 3-demethyl-O-acetyl-O-demethylADDA-homoarginine-microcystin- LR (MW, 1,022). The toxicities of the new microcystin homologs were not significantly different from those of microcystin-LR or demethylmicrocystin-LR.

Amino Acids↗
PubMed1989

A new procedure for the analysis and purification of naturally occurring anatoxin-a from the blue-green alga Anabaena flos-aquae.

Anabaena flos-aquae produces at least two neurotoxins termed anatoxin-a (ANTX-a) and -a(s) [ANTX-a(s)]. ANTX-a is a potent postsynaptic depolarizing neuromuscular blocking agent. Its structure is the secondary amine, 2-acetyl-9-azabicyclo[4.2.1]non-2-ene. Known isolation and analysis methods have not always given satisfactory results. Therefore, an improved procedure was developed for both isolation and analysis of ANTX-a which involves four steps; extraction, clean-up, separation and determination. Extraction was efficiently done with 0.05 M acetic acid, and a reversed phase. ODS (octadecylsilanized) and a cation exchanger (COOH) organosilans bonded to silica gels were applied to a clean-up step. Separation and determination were then performed using two TLC and HPLC chromatographies.

Bacterial Toxins↗
PubMed1989

Regional brain cholinesterase activity in rats injected intraperitoneally with anatoxin-a(s) or paraoxon.

Adult male Long-Evans rats were injected intraperitoneally with 1.5, 3.0 or 9.0 micrograms/kg of anatoxin-a(s) that had been extracted from laboratory-grown Anabaena flos-aquae NRC-525-17, 800 micrograms/kg of paraoxon, or a control solution. Blood, anterior spinal cord, and brain cerebellar, cortical, medullary, midbrain, hippocampal, hypothalamic, olfactory and striatal cholinesterase activity was determined in rats that died prior to 2 hours or were anesthetized and killed at 2 hours. Unlike paraoxon, anatoxin-a(s) did not cause detectable inhibition of cholinesterase in the central nervous system, but did cause inhibition of cholinesterase in blood, suggesting that anatoxin-a(s) is strictly a peripheral cholinesterase inhibitor.

Animals↗
PubMed1989

Algae intoxication in livestock and waterfowl.

Blue-green algae toxins include (1) hepatotoxic peptides that are known to be toxic to cattle, dogs, swine, waterfowl, and sometimes other species; (2) a nicotinic agonist neurotoxin that appears to be toxic to a wide range of animal species; (3) a peripheral-acting cholinesterase inhibitor that is very toxic to swine, birds, and dogs; (4) toxins that impair nervous transmission by blocking sodium channels in nerve cells; and (5) lipopolysaccharide endotoxins. This article provides current information on the mechanisms of action of the primary toxins recognized to date as well as on procedures important in the diagnosis and management of some of the more common cyanobacterial toxicoses in livestock and waterfowl.

Animals↗
PubMed1989

A model system for studying the bioavailability of intestinally administered microcystin-LR, a hepatotoxic peptide from the cyanobacterium Microcystis aeruginosa.

Sprague-Dawley rats were used to evaluate a model system for studying the hepatotoxicity caused by microcystin-LR (MCYST-LR), a toxin produced by the cyanobacterium (blue-green alga) Microcystis aeruginosa, and for evaluating the in vivo therapeutic potential of cholestyramine resin (CTR) which was found to bind the toxin in vitro. Female rats were treated with either toxin or an equivalent volume of the saline vehicle by direct administration into the lumen of an in situ isolated ileal loop. Male rats were dosed with toxin as described above, and then animals were dosed in the ileal loop with either cholestyramine resin (CTR, 50 mg/rat) or an equivalent vehicle. The survivors in both studies were killed six hours after dosing and hepatotoxicity was assessed by change in relative liver weights. In all groups given toxin alone, there was a significant increase in liver weight and males and females were equally susceptible. Liver weights of the toxin plus CTR treated rats were similar to those in vehicle-treated rats. When the toxin was administered into a similarly isolated jejunal loop, liver weight was significantly less than that found when an equivalent dose was administered into the ileal loop suggesting an intestinal site specificity for toxin absorption.

Animals↗
PubMed1989

Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin.

Water blooms formed by potentially toxic species of cyanobacteria are a common phenomenon in the Baltic Sea in late summer. Twenty-five cyanobacterial bloom samples were collected from open and coastal waters of the Baltic Sea during 1985 to 1987, and their toxicity was determined by mouse bioassay. All of 5 bloom samples from the southern Baltic Sea, 6 of 6 from the open northern Baltic Sea (Gulf of Finland), and 7 of 14 Finnish coastal samples were found to contain hepatotoxic cyanobacteria. Nodularia spumigena and Aphanizomenon flos-aquae occurred together in high amounts in blooms from the open-sea areas. In addition, coastal samples contained the species Anabaena lemmermannii, Microcystis aeruginosa, and Oscillatoria agardhii. Eighteen hepatotoxic N. spumigena cultures were isolated from water bloom and open-sea water samples. High-pressure liquid chromatographic analysis of both hepatotoxic bloom samples and Nodularia strains showed a single toxic fraction. The toxin concentrations of the blooms were less than or equal to 2.4 mg/g of freeze-dried material, and those of laboratory-grown cultures were 2.5 to 8.0 mg/g of freeze-dried cells. A single toxin was isolated from three N. spumigena-containing bloom samples and three N. spumigena laboratory isolates. Amino acid analysis and low- and high-resolution fast-atom bombardment mass spectroscopy indicated that the toxin from all of the sources was a cyclic pentapeptide (molecular weight, 824) containing glutamic acid, beta-methylaspartic acid, arginine, N-methyldehydrobutyrine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1988

Analysis and purification of toxic peptides from cyanobacteria by reversed-phase high-performance liquid chromatography.

A simple, rapid and reliable chemical analysis method for microcystins (cyanoginosins) has been studied. Three different mobile phases for high-performance liquid chromatography were selected and optimized. Also the adsorptive powers of three commercially available C18 cartridges were compared and the results successfully applied to the clean up of three of the toxins. Finally a total system for the analysis and isolation of microcystins was established.

Chromatography, High Pressure Liquid↗
PubMed1988

Improved method for purification of toxic peptides produced by cyanobacteria.

The improved method consists of ODS-silica gel extraction, and separation on silica gel and HPLC with UV (238 nm) detector. The method has been successfully applied to the isolation of toxic peptides from the Monroe and M-228 strains of Microcystis aeruginosa. This method reduces toxin extraction and separation time, and so enables a rapid isolation of peptide toxins from cyanobacteria.

Chromatography, Gel↗
PubMed1988

Comparison of effects of anatoxin-a(s) and paraoxon, physostigmine and pyridostigmine on mouse brain cholinesterase activity.

Anatoxin-a(s), an alkaloid neurotoxin from the freshwater cyanobacterium, Anabaena flos-aquae NRC-525-17, was compared to paraoxon, physostigmine and pyridostigmine for effects on brain cholinesterase after i.p. injection into Balb/c mice. The duration of clinical signs in mice injected with anatoxin-a(s) persisted longer than in mice given the carbamates and was comparable with that of paraoxon. Anatoxin-a(s) did not inhibit brain cholinesterase activity suggesting that this toxin is unable to cross the blood-brain barrier.

Animals↗
PubMed1986

Excretion of deoxynivalenol and its metabolite in milk, urine, and feces of lactating dairy cows.

Corn contaminated with deoxynivalenol was added to the diets of three dairy cows for 5 d and milk, urine, and 3 d following feeding of the diets. Dietary concentrations of deoxynivalenol averaged 66 mg/kg. Following exposure to deoxynivalenol, unconjugated deepoxydeoxynivalenol, a metabolite of deoxynivalenol, was present in milk at concentrations up to 26 ng/ml. Deoxynivalenol was not detected in the milk. Approximately 20% of the deoxynivalenol fed was recovered in the urine and feces in the unconjugated forms as deepoxydeoxynivalenol (96%) and deoxynivalenol (4%). After incubating urine with beta-glucuronidase, the concentration of unconjugated deepoxydeoxynivalenol increased by 7 to 15-fold whereas unconjugated deoxynivalenol increased 1.6 to 3-fold. Detectable concentrations of unconjugated deepoxydeoxynivalenol were found in urine and feces up to 72 h after the last oral exposure. Thus, urine and feces are the diagnostic specimens of choice for the determination of deoxynivalenol exposure in cows. Feeding deoxynivalenol-contaminated diets for 5 d did not alter feed intake or milk production nor were the milk concentrations of calcium, phosphorus, sodium, potassium, magnesium, or nitrogen altered.

Animals↗