Screening of boldenone and methylboldenone in bovine urine using disposable electrochemical immunosensors

Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone-bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG-horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3′,5,5′-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9 ± 4.3 pg ml⁻¹ for boldenone and 120.2 ± 8.2 pg ml⁻¹ for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC₅₀ = 1.0 ± 0.3 ng ml⁻¹ (n = 3, N = 3), R² = 0.969, R.S.D. = 9.6% for boldenone and 1.5 ± 0.3 ng ml⁻¹, 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of α-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.     

A comparative study of muscle force estimates using Huxley's and Hill's muscle model

Determination of muscle forces in individual muscles is often essential to assess optimal performance of human motion. Inverse dynamic methods based on the kinematics of the given motion and on the use of optimisation approach are the most widely used for muscle force estimation. The aim of this study was to estimate how the choice of muscle model influences predicted muscle forces. Huxley's (1957, Prog Biophys Biop Chem. 7: 255–318) and Hill's (1938, Proc R Soc B. 126: 136–195) muscle models were used for determination of muscle forces of two antagonistic muscles of the lower extremity during cycling. Huxley's model is a complex model that couples biochemical and physical processes with the microstructure of the muscle whereas the Hill's model is a phenomenological model. Muscle forces predicted by both models are within the same range. Huxley's model predicts more realistic patterns of muscle activation but it is computationally more demanding. Therefore, if the overall muscle forces are to be assessed, it is reasonable to use a simpler implementation based on Hill's model.     

A new species of Paracapillaria (Nematoda: Capillariidae) from the intestine of the toad Duttaphrynus melanostictus (Anura) from the Malayan Peninsula

A new species of parasitic nematode, Paracapillaria malayensis n. sp. (Capillariidae), is described from the small intestine of the toad Duttaphrynus melanostictus imported from the Malayan Peninsula to the Czech Republic. The new species differs from the only other congeneric species, Paracapillaria spratti, mainly in the shape and structure of the spicular proximal end (with a lobular rim), smaller eggs (45-51 x 21-24 microm), longer spicule (336 microm), and the number (37-38) of stichocytes in gravid females; whereas P. spratti parasitizes frogs of the Microhylidae in Papua New Guinea, P. malayensis is a parasite of Bufonidae in the Malayan Peninsula. Other Paracapillaria spp. are parasites of fishes, birds, or mammals and they mostly differ from P. malayensis in the structure of eggs and some other morphological features.     

Nitrous acid deamination of conformationally inverted aminodeoxyhexopyranoses

Nitrous acid deamination of 2-amino-1,6-anhydro-2-deoxy-β-D-glucopyranose (1) in the presence of weakly acidic, cation-exchange resin gave 1,6:2,3-dianhydro-β-D-mannopyranose (3) and 2,6-anhydro-D-mannose (6), characterized, respectively, as the 4-acetate of 3 and the per-O-acetylated reduction product of 6, namely 2,3,4,6- tetra-O-acetyl-1,5-anhydro-D-mannitol, obtained in the ratio of 7:13. Comparative deaminatior of the 4-O-benzyl derivative of 1 led to similar qualitative results. Deamination of 3-amino-1,6-anhydro-3-deoxy-β-D-glucopyranose gave 1,6:2,3- and 1,6:3,4-dianhydro-β-D-allopyranose (13 and 16), characterized as the corresponding acetates, obtained in the ratio of 31:69, as well as the corresponding p-toluenesulfonates. Deamination of 4-amino-1,6-anhydro-4-deoxy-β-D-glucopyranose and of its 2-O-benzyl derivative gave the corresponding 1,6:3,4-D-galacto dianhydrides as the only detectable products. 2,5-Anhydro-D-glucose, characterized as the 1,3,4,6-tetra-O- acetyl derivative of the corresponding anhydropolyol, was obtained in 39% yield from the same deamination reaction performed on 2-amino-1,6-anhydro-2-deoxy-β-D- mannopyranose (24). In 90% acetic acid, the nitrous acid deamination of 24, followed by per-O-acetylation, gave only 1,3-4-tri-O-acetyl-2,5-anhydro-α-D-glucoseptanose. In the case of 1,6-anhydro-3,4-dideoxy-3,4-epimino-β-D-altropyranose, only the corresponding glycosene was formed, namely, 1,6-anhydro-3,4-dideoxy-β-D-threo--hex-3-enopyranose.     

Effect of ischemia and reperfusion on protein oxidation in isolated rabbit hearts

Reactive oxygen species and other oxidants are involved in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. The present study investigated the oxidative modification of cardiac proteins in isolated Langendorff-perfused rabbit hearts subjected to 15 min normothermic ischemia followed by 10 min reperfusion. Reperfusion under these conditions resulted in only 61.8+/-2.7 % recovery of developed pressure relative to preischemic values and this mechanical dysfunction was accompanied by oxidative damage to cardiac proteins. The total sulfhydryl group content was significantly reduced in both ventricle homogenates and mitochondria isolated from stunned hearts. Fluorescence measurements revealed enhanced formation of bityrosines and conjugates of lipid peroxidation-end products with proteins in cardiac homogenates, whereas these parameters were unchanged in the mitochondrial fraction. Reperfusion did not alter protein surface hydrophobicity, as detected by a fluorescent probe 1-anilino-8-naphthalenesulfonate. Our results indicate that oxidation of proteins in mitochondria and possibly in other intracellular structures occurs during cardiac reperfusion and might contribute to ischemia-reperfusion injury.     

Bromide transfer through mother's milk and its impact on the suckling rat

Effects of a high bromide intake in lactating rats on the performance of the dams and on the prosperity of their young were studied. In the dams, two marked consequences undoubtedly caused by high bromide intake were observed: stagnation in the extent of diet and water consumption in the course of the lactation period, and a conspicuous drop in the production rate of mother's milk. A very high intake of bromide in the mothers in the course of the nursing period (about 220 mg Br⁻/d per dam) also caused a marked decrease in the body weight increments in their suckling young. Only about one-half of these young survived and their general condition was very poor. It is suggested that one of the possible reasons for the observed marked decrease in the production of mother's milk in dams with high bromide intake could be a decreased stimulation of the mammary glands as a consequence of reduced consumption of mother's milk by the suckling. Bromide ions ingested by the dams easily moved into the rat milk. Via mother's milk, bromide was transferred in a large extent to the suckling. The amount of bromide in mother's milk depended on the bromide concentration in the drinking water taken by the dams. With the addition of 5 g bromide per liter (providing the mean daily bromide dose of 220 mg), bromide ions replaced about 54% of the chloride in the milk. A rise in the concentration of both halogens caused also an increase in the concentration of sodium in mother's milk. The exact mechanism(s) of bromide interference with postnatal developmental processes in the young remain(s) unclear. Presented in part at the, 4th International Symposium on Trace Elements in Human: New Perspectives held in Athens (Greece) on 9–11 October 2003.     

Pseudomonas C12B, an SDS degrading strain, harbours a plasmid coding for degradation of medium chain length n-alkanes

Pseudomonas C12B is able to degrade alkyl sulfates, alkylbenzene sulfonates, and linear alkanes and alkenes. Mitomycin C curing experiments and conjugation experiments demonstrated that the ability to utilize n-alkanes (C₉–C₁₂) and n-alkenes (C₁₀ and C₁₂) of medium chain length was plasmid-encoded. The plasmid was designated pDEC. Its size was estimated at several hundreds kb according to mobility in agarose gels. The plasmid did not confer resistance to the antibiotics tested. Analysis of alkylsulfatases P1 and P2 in original and cured strains confirmed that both enzymes are encoded by the chromosome. The ability of Pseudomonas C12B to utilize alkylbenzene sulfonates also appears to be encoded by the chromosome. pDEC could be transferred only to cured derivatives of Pseudomonas C12B, but not to strains of P. aeruginosa, P. putida, or Acinetobacter sp. Cured derivatives of Pseudomonas C12B could not serve as hosts for the broad host range plasmid CAM–OCT. The enzyme system encoded by the putative dec genes present on plasmid pDEC differs from the system coded by the alk genes of plasmid OCT in the size range of hydrocarbons preferentially used.     

Structure of Micrococcus cryophilus after freeze-etching

Summary Principle. Nitrate reductases A and B use methyl viologen as an electron donor. This oxidation-reduction indicator is reduced, in anaerobic conditions, by an excess of dithionite. The nitrite produced is estimated by colorimetry. Enzyme A. The activity is stable for at least the first 30 min of the reaction and it is proportional to enzyme concentration. Enzyme B. The activity decreases rapidly and continuously from the start of the reaction. Conclusion. The colorimetric method can be used to measure the activity of enzyme A. But it is inapplicable to enzyme B.

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Les nitrate-réductases bactériennesVII. Mesure de l'activité des enzymes A et B par une méthode colorimétrique

Résumé Principe. Les nitrate-réductases A et B utilisent le méthyl-viologène comme donneur d'électrons. Cet indicateur d'oxydoréduction est réduit, en anaérobiose, par un excès d'hydrosulfite. Le nitrite produit est dosé par colorimétrie. Enzyme A. L'activité demeure constante pendant 30 min au moins après le début de la réaction et elle varie proportionnellement à la concentration d'enzyme. Enzyme B. L'activité diminue rapidement et de façon continue dès le début de la réaction. Conclusion. La méthode colorimétrique perment de mesurer l'activité de l'enzyme A. Par contre, elle est inapplicable à l'enzyme B.