Melatonin promotes sleep-like state in zebrafish.

The sleep-promoting effect of the pineal hormone melatonin in humans is known for decades. However, the mechanisms of this phenomenon remain obscure, mainly due to lack of a simple, genetically tractable, animal model. We now report that melatonin promotes sleep-like state in a diurnal lower vertebrate, zebrafish (Danio rerio), and this effect is mediated through activation of specific melatonin membrane receptors. Furthermore, our data show that the sleep-like state in zebrafish has fundamental similarities with sleep in mammals, including characteristic postures, elevated arousal threshold to sensory stimulation and a compensatory rest rebound following rest deprivation, and can be induced by conventional hypnotics, diazepam and sodium pentobarbital. Collectively, these data indicate that melatonin is evolutionary conserved sleep-promoting agent in diurnal species and suggest that zebrafish provide an efficient animal model for studying the molecular mechanisms of sleep regulation and for screening new types of hypnotic medications.

[Prospects for use of hapten-heterologous conjugates in immunoassay of thyroid hormones]. / Vozmozhnosti ispol'zovaniia gapten-geterologichnykh kon''iugatov v immunoanalize tiroidnykh gormonov.

The effect of hapten heterology on the characteristics of indirect ELISA methods for determination of thyroxine and triiodothyronine using monoclonal antibodies was studied. The use of the heterologous triiodothyronine-bovine serum albumin conjugate in immunoassays for thyroxine improved the sensitivity of these assays twofold and reduced the cross-reactivity with triiodothyronine from 1 to 0.5% as compared to the homologous variant. By contrast, the heterology in the immunoassays for triiodothyronine appeared inadequate. It was shown that the specificity and sensitivity of hapten immunoassays can be modulated by altering the chemical structure of the hapten-label conjugate, which is most evident in experiments with monoclonal antibodies.

[Determination of phenobarbital by solid-phase immunoenzyme analysis. Choice of optimal strategy]. / Opredelenie fenobarbitala metodami tverdofaznogo immunofermentnogo analiza. Vybor optimal'noi strategii.

Two variants (direct and indirect) of enzyme linked immunosorbent assay (ELISA) of phenobarbital are compared. Both techniques were developed on the basis of the same monoclonal antibodies, and horse radish peroxidase was used as the label in both cases. When microtitration plates are used as the solid phase, indirect ELISA, in which phenobarbital of the sample competes with phenobarbital sorbed on plates in the form of a conjugate with protein for the binding with peroxidase-labeled antiphenobarbital antibodies, is preferable. In indirect ELISA, the sample volume was 5 microliters, the time of assay was 40 min, the variability coefficient was < 8%.

ELISA screening of monoclonal antibodies to haptens: influence of the chemical structure of hapten-protein conjugates.

An analysis has been made of the influence of the chemical structure of hapten-protein conjugates on ELISA based screening monoclonal antibodies to haptens. Evidence has been obtained that the size of haptens should be taken into account. For haptens with a relatively large molecular weight, one can use a conjugate with a carrier protein differing from the immunogen. For small haptens, the screening should be conducted with a conjugate differing from the immunogen with respect to both the carrier protein and the chemical linkage between the hapten and the protein. In addition, the hapten-protein coupling should involves different amino acid residues.

[Preparation of monoclonal antibodies to phenobarbital]. / Poluchenie monoklonal'nykh antitel k fenobarbitalu.

Monoclonal antibodies to phenobarbital have been developed. Spleen cells of BALB/c mouse immunized with conjugate keyhole limpet haemocyanin and phenobarbital were fused with P3-X63-Ag8.653 mouse myeloma cells. Three monoclonal antibodies, selected by indirect ELISA, were produced in mouse ascite fluids, purified and analyzed. Antibody 1A9 was selected for use in immunoassay, its association constant being 1.6 x 10(9) M-1.

[Determination of phenobarbital by polarized fluoroimmunoanalysis. The effect of immunogen structure and tracer on specificity and detection limit]. / Opredelenie fenobarbitala metodom poliarizatsionnogo fluoroimmunoanaliza. Vliianie struktury immunogena i treisera na spetsifichnost' i predel detektsii.

The influence of the immunogen's tracer's structure on properties of antibodies to phenobarbital was studied by the polarisation fluoroimmunoassay method. It was shown that in homogeneous method of polarisation fluoroimmunoassay the titer of antibodies to phenobarbital (1/1000-1/10,000) is affected by the position of linker binding the antigen with carrier-protein and by the structure of the linker. The affinity constant ((1-4) x 10(9) M-1), cross-reactivity, limit of the detection (0.6-2.6 micrograms/ml) phenobarbital with different combinations of antibodies and tracers were calculated. The antisera grew the more heterogeneous in affinity and specificity, the longer became the linker. The more heterogeneous antibodies are preferable in the class specific assay of barbiturates.

Fast filtration enzyme immunoassay for haptens.

An immunometric method for determination of hapten concentration in fluids has been developed. High-affinity hapten-specific enzyme-labeled monoclonal antibodies are mixed with a sample containing hapten, then the mixture is filtered through a membrane with immobilized hapten. The level of enzyme activity retained by the membrane is inversely proportional to the concentration of hapten in a sample. The assay has been developed for theophylline, digoxin and phenobarbital. The coefficient of variation is less than 5% and the test takes about 2 min.

[Effect of enomelanin on erythrocyte hemolysis induced by free-radical reactions and other factors]. / Vliianie énomelanina na gemoliz éritrotsitov, vyzyvaemyi svobodnoradikal'nymi reaktsiiami i drugimi faktorami.

Grapes-isolated melanin (enomelanin) was shown to inhibit red cell haemolysis induced by Fenton reagent, photoactivated chlorpromazine and antierythrocyte antibodies. Enomelanin failed to produce any effect on osmotic hemolysis.

[Preparation of antibodies against o-aminoazotoluene using chemically and enzymatically synthesized conjugated antigens]. / Poluchenie antitel k o-aminoazotoluolu s pomoshch'iu sintezirovannykh khimicheskim i fermentativnym metodami kon''iugirovannykh antigenov.

Two methods are developed for synthesis of conjugated antigens to o-aminoazotoluene (o-AAT)-albumin: the enzymatic method--using horseradish peroxidase (HP) and the chemical one--diazomethod. A possible mechanism is suggested for the covalent binding of o-AAT to albumin which is catalyzed by HP. Antibodies to o-AAT are obtained by immunization of animals. An essential difference is established for antigenic o-AAT determinants in conjugates synthesized by the enzymatic and chemical methods.

[Synthesis, physico-chemical and antigenic properties of the conjugated antigens of xanthine- and theophylline proteins]. / Sintez, fiziko-khimicheskie i antigennye svoistva kon''iugirovannykh antigenov ksantin- i teofillin-belok.

Conjugated antigens of xanthine- and theophylline-protein were synthesized by the reaction of diazosalts of 8-aminoxanthine and 8-aminotheophylline with bovine serum albumin. UV-spectra of the synthesized preparations were studied and content of xanthine and theophylline, covalently bound with the substances, was estimated. Immunization of animals with the conjugates was found to cause the occurrence of antibodies in blood serum, which bound specifically various derivatives of purine.