Previous studies underestimate BMAA concentrations in cycad flour.

The traditional diet of the Chamorro people of Guam has high concentrations of the neurotoxin BMAA, beta-methyl-amino-L-alanine, in cycad tortillas and from animals that feed on cycad seeds. We measured BMAA concentration in washed cycad flour and compared different extraction methods used by previous researchers in order to determine how much BMAA may have been unaccounted for in prior research. Samples were analyzed with AQC precolumn derivatization using HPLC-FD detection and verified with UPLC-UV, UPLC-MS, and triple quadrupole LC/MS/MS. Although previous workers had studied only the free amino acid component of BMAA in washed cycad flour, we detected significant levels of protein-associated BMAA in washed cycad flour. These data support a link between ALS/PDC and exposure to BMAA.

Cyanobacteria and BMAA: possible linkage with avian vacuolar myelinopathy (AVM) in the south-eastern United States.

Avian vacuolar myelinopathy (AVM) is a neurological disease that produces uncoordinated behavior in affected birds in wetland ecosystems of the south-eastern United States. Feeding and sentinel trials, field surveys, and genetic studies have implicated the introduced flowering plant species Hydrilla verticillata (Hydrocharitaceae) and an associated epiphytic cyanobacterial species (Order Stigonematales) as a causal link to AVM. All five morphotypes of cyanobacteria have been shown to produce the neurotoxic amino acid BMAA, including cyanobacteria of the Stigonematales that are epiphytic on Hydrilla verticillata. If biomagnification of BMAA occurs in these wetland ecosystems, as has been observed in the Guam ecosystem, then the consumption of fish (e.g. shad and herring) and waterfowl (e.g. Canada geese and mallards) from AVM-confirmed reservoirs in Arkansas, Texas, Georgia, North Carolina and South Carolina could represent a significant human health risk.

Presence of the neurotoxic amino acids beta-N-methylamino-L-alanine (BMAA) and 2,4-diamino-butyric acid (DAB) in shallow springs from the Gobi Desert.

The Gobi Desert in Mongolia, home to the critically endangered Gobi bear (Ursus arctos isabellinus), has few water resources for the animals that inhabit this environment. The majority of these water resources are shallow, small bodies of water, from approximately 30 cm to 3 m in diameter. Due to the harsh nature of the Gobi Desert environment, such pools of water are crucial resources for wildlife inhabiting the area and little information is currently available on the presence of organisms, including cyanobacteria, and the toxins they produce within these waters. Drinking water sources and small pools within the Gobi Desert were sampled on two separate occasions in October 2008 and April-May 2009. Samples were assessed for the presence of cyanobacteria; subsamples were taken for the analysis of beta-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). According to LC-MS/MS analyses, both of these neurotoxic amino acids were present in both years and BMAA was present when cyanobacteria were major components of the pools. The results indicate that assessment of cyanotoxins to organisms that live in desert environments is warranted.

Cyanobacteria and BMAA exposure from desert dust: a possible link to sporadic ALS among Gulf War veterans.

Veterans of the 1990-1991 Gulf War have been reported to have an increased incidence of amyotrophic lateral sclerosis (ALS) compared to personnel who were not deployed. An excess of ALS cases was diagnosed in Gulf War veterans younger than 45 years of age. Increased ALS among Gulf War veterans appears to be an outbreak time-limited to the decade following the Gulf War. Seeking to identify biologically plausible environmental exposures, we have focused on inhalation of cyanobacteria and cyanotoxins carried by dust in the Gulf region, particularly Qatar. Cyanobacterial crusts and mats are widespread in the deserts of Qatar, occupying up to 56% of the available area in some microhabitats. These cyanobacterial crusts, which help bind the desert sands, are dormant throughout most of the year, but during brief spring rains actively photosynthesize. When disturbed by vehicular traffic or other military activities, the dried crusts and mats can produce significant dust. Using HPLC/FD, an amino acid analyzer, UPLC/MS, and triple quadrupole LC/MS/MS we find that the dried crusts and mats contain neurotoxic cyanobacterial toxins, including beta-N-methylamino-L-alanine (BMAA) and 2,4 diaminobutyric acid (DAB). If dust containing cyanobacteria is inhaled, significant exposure to BMAA and other cyanotoxins may occur. We suggest that inhalation of BMAA, DAB, and other aerosolized cyanotoxins may constitute a significant risk factor for the development of ALS and other neurodegenerative diseases.

Possible therapy for ALS based on the cyanobacteria/BMAA hypothesis.

Although the cyanobacteria/BMAA hypothesis of the cause of ALS and other age-related neurodegenerative diseases remains to be proven, it is not too early to ask whether treatment would be possible if the hypothesis were correct. This paper reviews the possible ways that chronic BMAA neurotoxicity could be prevented or treated.

Resolution of DL-2-aminosuberic acid via protease-catalyzed ester hydrolysis.

Papain-catalyzed regioselective cleavage of alpha-methyl ester in Z-DL-Asu(OMe)-OMe leads to Z-L-Asu(OMe)-OH and Z-D-Asu(OMe)-OMe. Subsequent saponifications yield Z-L-Asu-OH and Z-D-Asu-OH. The enzymatic alpha-ester hydrolysis was also achieved by subtilisin BPN' in organic solvent with low water content.

Halophilic class I aldolase and glyceraldehyde-3-phosphate dehydrogenase: some salt-dependent structural features.

Aldolase and glyceraldehyde-3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis are stable only in high concentrations of KCl present within the physiological environment. Data concerning the structural changes in the two enzymes as a result of lowering of salt concentration and changes in pH were obtained by monitoring the intrinsic protein fluorescence in the presence of quenchers. When the KCl concentrations were lowered below 2 M or in the presence of 6 M guanidine hydrochloride, the emission maximum shifted to a longer wavelength, indicating enhanced exposure of tryptophyl residues to the solvent. The spectral characteristics of the two proteins in guanidine hydrochloride and 0.4 M KCl were identical. However, these denatured states appear to be different than those observed after acid denaturation. Further perturbation of fluorescence was observed due to I-, and application of the Stern-Volmer law showed that the total fluorescence was available to the quenchers only in 0.4 M KCl solutions. The unfolding of proteins in 0.4 M KCl was a gradual process which was accompanied by a time-dependent loss in enzyme activity. The activity loss was complete within 30 min for aldolase whereas in the case of GAPDH nearly 3 h was required for the destruction of activity. For both enzymes, inactivation and protein denaturation were strongly correlated. The data on activity and thermostability measurements of the two enzymes in varying concentrations of KCl and potassium phosphate revealed that though both proteins are halophilic, the forces in the maintenance of their stability could be different.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride.

Oxidatively modified proteins have been implicated in a variety of physiologic and pathologic processes. Oxidative modification typically causes inactivation of enzymes and also the introduction of carbonyl groups into amino acid side chains of the protein. We describe a method to quantify oxidatively modified proteins through reduction of these carbonyl groups with tritiated borohydride. The technique was applied to purified, oxidatively modified glutamine synthetase and to bronchoalveolar lavage fluid from dogs and from humans. Since the protein content of lung lavage fluid is low, a very sensitive method was required to measure the oxidized residues. Reduction of the carbonyl group generated during oxidation of proteins with tritiated borohydride provided excellent sensitivity. Incorporation of tritium was directly proportional to the amount of protein with a range from 10 to 1000 micrograms. Should moieties other than amino acids be labeled, they are easily removed by rapid benchtop hydrolysis of the protein followed by chromatography on Dowex 50.

Topographical distribution of neurochemical changes in Alzheimer's disease.

Biochemical indices of cortical nerve cells affected in Alzheimer's disease have been proposed (excitatory dicarboxylic amino acid, EDAA, sodium-dependent carrier; phosphate-activated glutaminase activity; serotonin type 2 recognition site; somatostatin-like immunoreactivity). These and the content of EDAAs and two related amino acids, and choline acetyltransferase (ChAT) activity have been measured in up to 13 areas of cerebral cortex and the cerebellar cortex from 16 patients with Alzheimer's disease and 17 controls. Reduction of the index of the serotonin recognition site, somatostatin content and another biochemical index of interneurones coincide and indicate a rather unexpected focal loss of such neurones from the parietal lobe. No unequivocal measure of the integrity of pyramidal neurones could be established as the content of no amino acid was reduced, the index of the EDAA carrier showed evidence of change in few brain regions and glutaminase activity was subject to unexplained variability. ChAT activity alone closely paralleled a previous report of the distribution of morphological degeneration. The results are discussed in relation to therapy and positron emission tomography.

[Dicarboxylic amino acids in brain proteins under conditions of sensitization with brain antigen]. / Dikarbonovye aminokisloty v belkakh mozga v usloviiakh sensivilizatsii mozgovym antigenom.

Under conditions of the organism sensibilization with brain antigen there occur changes in amino acid composition of the brain protein fractions. The ratio of negatively charged amino acids to positively charged ones in sharply shifted towards predominance of the negative charge. A conclusion is made that such a brain protein modification activates acid peptide hydrolases and intensifies the sensibility of the proteins to these acid proteinases.

Lysine-derived cross-links in the egg shell membrane.

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB3H4 reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.

Identification and quantification of alpha-amino adipic acid in bovine dentine phosphoprotein.

A unique phosphoprotein, which contains an uncommon amino acid, alpha-amino adipic acid (alpha-AAA), was isolated from unerupted bovine teeth by extensive EDTA demineralization. The alpha-AAA in the protein hydrolysates was identified based on the elution time on an amino acid analyzer using the sodium and lithium citrate buffer in a dual column system, and its identity was confirmed by comparisons of its DNS derivative with that of authentic alpha-AAA. This result suggests that the lysine residues in the phosphoprotein may be deaminated by an amine oxidase into allysine and further oxidized to alpha-AAA.

Sequential grouping of nonhistone chromosomal protein from pig thymus.

1. To establish a practical method to prepare nonhistone chromosomal proteins (NHP's) in substantial quantity, a sequential group fractionation procedure for NHP's from pig thymus chromatin has been developed by a combination of methods based on the intrinsic properties, such as binding force in chromatin, molecular weight, and acidity and/or basicity of the protein components. 2. The loosely bound NHP's in chromatin have been grouped into 11 subfractions, and tightly bound ones into 6 subfractions, 2 of which were histones. The loosely bound NHP's were chiefly acidic to neutral in contrast with the tightly bound components, which were basic. In addition, the NHP's of high molecular weight were in general acidic to neutral, while the lower molecular weight components were mainly basic. 3. The present method is applicable to the preparation of representative fractions of NHP on the scale of 50--400 mg from 1 kg of pig thymus.

[Changes in immune reactivity of the organism and content of monoaminodicarboxylic acid and their derivatives in brain and spleen]. / Izmenenie immunologicheskoi reaktivnosti organizma i soderzhanie monoaminodikarbonovykh kislot i ikh proizvodnykh v golovnom mozge i selezenke.

It is established in experiments with mice that the contrary effects on the immune system caused different changes in the level of monoaminodicarboxylic acids and their derivatives in the brain and spleen structures. While immunostimulation was accompanied by an increase in monoaminocarboxylic acids, immunodepression, on the contrary, was characterized by a decrease in their contents up to the level, which is lower than the initial one. The main task of the investigation was to find out if this correlation is of causal character, which might be indicative of the significance of maintaining the definite level of monoaminodicarboxylic acids and their derivatives in the brain immunocompetent organs for normal functioning of the immune system.

Isolation and nature of intracellular alpha-aminoadipic acid-containing peptides from Paecilomyces persicinus P-10.

Small intracellular peptides containing alpha-aminoadipic acid, cysteine, and a valine moiety were obtained from mycelia of Paecilomyces persicinus P-10 by ethanol or trichloroacetic acid extraction. After performic acid oxidation and ion-exchange chromatography, analysis of the peptide fractions by two-dimensional thin-layer electrophoresis and chromatography revealed the presence of three related peptides, as sulfonic acid derivatives, each containing alpha-aminoadipic acid. Each peptide was isolated in chromatographically pure form by semipreparative thin-layer electrophoresis and chromatography. The purified peptides were subjected to differential hydrolysis, dansylation, and combined dansylation-phenylisothiocyanate sequence analysis. Based on these studies, the structures of the isolated peptides were determined to be (i) glycl-delta-(alpha-aminoadipyl)-cysteinyl-beta-hydroxyvaline, (ii) glycyl-delta-(alpha-aminoadipyl)-cysteinylvaline, and (iii) delta-(alpha-aminoadipyl)-cysteinylvaline. The peptides isolated from Paecilomyces are similar to the alpha-aminoadipic acid-cysteine-valine moiety complex peptides isolated from Cephalosporium.

Meso-alpha,epsilon-diaminopimelate D-dehydrogenase: distribution and the reaction product.

A high activity of meso-alpha-epsilon-diaminopimelate dehydrogenase was found in extracts of Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, and Proteus vulgaris among bacteria tested. B. sphaericus IFO 3525, in which the enzyme is most abundant, was chosen to study the enzyme reaction. The enzyme was not induced by the addition of meso-alpha-epsilon-diaminopimelate to the growth medium. The reaction product was isolated and identified as alpha-amino-epsilon-ketopimelate by a comparison of the properties of its 2,4-dinitrophenylhydrazone with those of an authentic sample in silica gel thin-layer chromatography, absorption, infrared and proton nuclear magnetic resonance spectrometry, and elemental analyses. The alpha-amino-epsilon-ketopimelate formed enzymatically was decarboxylated by H2O2 to yield L-alpha-aminoadipate. This suggests that the amino group with D-configuration in the substrate is oxidatively deaminated; the enzyme is a D-amino acid dehydrogenase. L-alpha-Amino-epsilon-ketopimelate undergoes spontaneous dehydration to the cyclic delta1-piperideine-2,6-dicarboxylate. The enzyme reaction is reversible, and meso-alpha-epsilon-diaminopimelate was formed in the reductive amination of L-alpha-epsilon-ketopimelate.

Lysyl-derived aldehydes in outer membrane proteins of Escherichia coli.

The major outer membrane proteins from Escherichia coli K-12 are modified to contain alpha-aminoadipic acid delta-semialdehyde (allysine). The allysine was found to be derived from lysine and it was identified by derivatizing it to chloronorleucine by reduction, alpha-aminoadipic acid by oxidation, and to alpha,epsilon-diaminopimelic acid by reacting it with CN- and NH3. The alpha-aminoadipic acid was identified by mass spectrometry. Two major outer membrane proteins were found to possess allysine, a modified lysine characteristically found to connective tissue.