A sensitive biosensor for the fluorescence detection of the acetylcholinesterase reaction system based on carbon dots.

The carbon dots (C-dots) with high fluorescence quantum yield were prepared using hydrothermal method. C-dots have been adopted as probes for the fluorescence turn-off detection of H2O2 based on the special sensibility for the hydroxyl radical. And then the biosensors for the detection of substrate and enzymes activities were established in the acetylcholinesterase reaction system, which were related to the production of H2O2. Specifically, the proposed fluorescent biosensor was successfully applied to detect the concentration of choline (in the range from 0.025 to 50 µM) and acetylcholine (in the range from 0.050 to 50 µM), and the activity of choline oxidase (in the range from 1 to 75 U/L) and acetylcholinesterase (1 to 80 U/L). These results showed a sensitive, universal, nontoxic and eco-friendly detecting technique has been developed.

A sequential enzymatic microreactor system for ethanol detection of gasohol mixtures.

A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 +/- 2.3% and 63.2 +/- 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1-30 % v/v (10.0-238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034-0.087 g ethanol/L working with the on-line dilution integrated to the biosensor FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.

Polyporus s. str. in southern South America: isoenzyme analysis.

Forty-two dikaryotic and 42 monokaryotic isolates, and 34 pairings were examined by horizontal polyacrylamide gel electrophoresis (PAGE) for six enzymatic activities, viz. EST, 6PGD, IDH, MDH, SHDH and SOD. 44 bands were analysed. Numerical analysis of the isoenzymatic patterns was undertaken and compared with those from morphological characters. The analysis of six enzymatic systems showed the existence of four monomorphic systems (IDH, MDH, SHDH and SOD). The sterease system (EST) appears to be polymorphic in Polyporus ciliatus and in populations of P. tenuiculus from Argentina, being monomorphic in the remaining species studied. The 6PGD system is polymorphic in P. tucumanensis and monomorphic in the other species. Predominance of monomorphic enzymes and a clear distribution of the electromorphs among the species, indicates that isoenzymatic analysis is a good taxonomic tool within Polyporus. The low intraspecific variability allowed the use of interspecific differences to separate species. Numerical analysis showed a good correlation between morphological and molecular characters. In the isoenzymatic phenogram the similarity index is high only among very close species, showing a stressed separation of species.

Specialized metabolism and biochemical suppression during aestivation of the extant South American lungfish--Lepidosiren paradoxa.

Lepidosiren paradoxa (pirambóia) is the single representative of Dipnoan (lungfish) in South America. This species is considered a living fossil, in spite of some reports describing this fish as having a very specialized life style. It aestivates during the dry season, and has developed metabolic adaptations to cope with both flooding and drought. The literature describing its tissue ultra-structure shows high glycogen stored in the muscle, suggesting a strong dependence on anaerobic glycolysis. The present paper reports tissue enzyme levels of LDH, MDH, and CS, and isozymic tissue distribution of LDH, MDH, ADH, PGI, SOD, and PGM of 7 aestivating specimens from Lago do Canteiro in the Amazonas River. Animals were caught while burrowed in mud during the aestivation period. Our findings reveal high anaerobic capacity of both skeletal and heart muscles, even during the aestivation period, when enzymes showed suppressed levels compared to those of non-aestivating animals (data from the literature). Isozymic patterns suggest loss of duplicate condition in most analyzed loci, a characteristic that occurs mainly in higher vertebrate categories. These data indicate that, compared to the fish group, lungfish may be considered advanced, despite retaining primitive morphological characters.

Choline oxidase chemiluminescent assay, after removal of eserine from medium, of acetylcholine released in vitro from brain slices.

A chemiluminescent method has been used recently for the determination of acetylcholine with limitations such as the presence of a cholinesterase inhibitor in the incubation medium, which is indispensable for the study of acetylcholine release by various agents. A modified procedure is presented in which the cholinesterase inhibitor eserine (physostigmine) is extracted from the medium. The results showed complete recovery when labelled acetylcholine was used. This modified procedure was used to determine the release of acetylcholine evoked by tityustoxin and ouabain. The results were comparable to those obtained by bioassay using a strip of guinea pig ileum.

Further isoenzymatic studies on Trypanosoma cruzi stocks from Brazil, Colombia and Chile.

Eighteen stocks of T. cruzi from Brazil, Colombia and Chile were compared by five isoenzyme patterns of isoelectrofocusing with the three principal Brazilian zymodemes (Z1, Z2, Z3). The two main Groups I and II found confirmed the zymodemes 1 and 2. However, in some cases new isoenzyme types of the enzymes GPI and PGM in the Groups I and II have been seen. Stocks typed as Z3 did not show homogeneous enzyme patterns. A clear differentiation from the Z1 and Z2 or an association with a third group was not possible. The results of Chilean stocks showed the predominance of the Group II in the domestic cycle of T. cruzi, whereas the results of the Colombian stocks typed as Group I pointed out that in countries north of the Amazon basin only Group I of T. cruzi exists.

The microdistribution of isoenzymic strains of Trypanosoma cruzi in southern Bolivia; new isoenzyme profiles and further arguments against Mendelian sexuality.

132 Trypanosoma cruzi stocks were collected in southern Bolivia (99 stocks in Tupiza, 33 in Tarija), and were characterized using five enzymes (six loci). From these 132 stocks, a sample of 21 was studied using 10 enzymes (12 loci) to establish the genetic distances between them. Only five different isoenzymic strains were registered among the 132 stocks: the taxonomic status of these strains is discussed. The distribution of the strains indicated that a Founder effect was not a constant fact at the level of the house and of the suburb, but that a Founder effect was more apparent for greater geographical distances. All strains were transmitted sympatrically by the same vector Triatoma infestans. Genotype frequencies demonstrated the lack of Mendelian sexuality among stocks of T. cruzi from southern Bolivia, confirming our previous results.

Human alcohol dehydrogenase ADH1, ADH2 and ADH3 loci in a mixed population of Bahia, Brazil.

1. The three structural gene loci of human alcohol dehydrogenase have been studied in liver, jejunum and lung from 300 newborns in a triracially mixed population of Bahia, Brazil. 2. The frequency of the ADH23 allele was 0-1392, suggesting that the ADH23 allele is less frequent in Negroes. 3. A new ADH2 variant was identified. The electrophoretic pattern was interpreted as due to a new allele which is provisionally called ADH2Bahia. 4. By electrophoretic classification the 'atypical' variant was found in 2-8% of the sample. A question is raised regarding the ancestral origin of the 'atypical' variant in the population. Because this variant is common in Japanese it may have reached the present day population of Bahia through their American Indian ancestors. 5. Subjective estimation of the proportions of beta chains by giving scores to the liver isozymes alphaalpha, alphabeta and betabeta showed a clear relationship between the fetal weight and the beta chain activity. 6. The proportion of beta chains in the liver is significantly less when there is no enzyme activity in the lung, indicating some synchronous 'turning on' mechanism for alcohol dehydrogenase synthesis in both tissues.