Rapid Detection of Femtogram Amounts of Protein by Gel-Free Immunoblot.

The article presents a new method of immunoblotting for simple, rapid, and highly sensitive detection of proteins. Electrophoretic separation of sample is carried out under non-denaturing conditions in a thin conductive layer between cellulose membranes without polyacrylamide gel. The membrane surface is preliminarily modified with azidophenyl groups to photochemically immobilize proteins in situ. For visualization of protein bands, the membranes are treated with magnetic beads coated with specific antibodies, unbound particles are then removed with a magnet. The detection limit in the model system with biotinylated BSA and magnetic beads coated with streptavidin reaches 10 fg or about 105 molecules, while the total blotting time does not exceed 5 min. The method was applied for detection of IgA in a sample of human exhaled air. The method can be used for the analysis of various complex biological samples containing low amounts of the analyte.

[Study of bioavailability of different forms of synthetic beta-carotene in volunteers]. / Izuchenie biodostupnosti razlichnykh form sinteticheskogo beta-karotina na dobrovol'tsakh.

The concentration of beta-carotene analyzed in blood serum of 30 healthy men and women aged 17-50 years by the method of HPLC in May-June 1990 was in range 3.3-29.5 and in average 12.0 +/- 1.2 micrograms/100 ml. Concentration of carotene determined by Bessay's spectrophotometric method as sum of carotenoids composed 58.0-215.0 and in average 120.5 +/- 7.5 micrograms/100 ml. This level of carotenoids is higher by 10 times than level of beta-carotene detected by HPLC. But the levels of carotenes in blood serum detected by two methods are in positive correlation (r = 0.8). Single intake by volunteers 25 mg of synthetic beta-carotene in three different forms (as cyclodextrin derivation, or 30% microcrystalic suspension in vegetable oil, or 10% water-soluble form produced by Hoffman-La Roche company) caused increasing of beta-carotene concentration in serum with maximum within 24-48 hours. Bioavailability of cyclodextrin derivation of beta-carotene, determined by absolute increasing of beta-carotene concentration in serum, was lowest than that of other forms. Bioavailability of beta-carotene as 30% microcrystalic suspension in vegetable oil or 10% water-soluble form was practically the same. Intake of beta-carotene did not effect on level of retinol in serum which was in normal range.

[Bioavailability and pharmacodynamics of various forms of synthetic beta-carotene in volunteers]. / Izuchenie biodostupnosti i farmakodinamiki razlichnykh form sinteticheskogo beta-karotina na dobrovol'tsakh.

The biological availability of artificial beta-carotene as a water-soluble versus oily formulation based on cyclodextrin (Cyclocar tablets) was studied on volunteers given a single dose of 25 mg. The concentrations of beta-carotene and major carotenoids were measured in the blood serum during the experiment by high performance liquid chromatography. The maximum content of beta-carotene in the serum was attained 24-30 and 30-48 hrs after oily formulations and Cyclocar and were 48.0 +/- 7.7 and 28.1 +/- 3.6 mg/dl, respectively. The rate of beta-carotene utilization from Cyclocar was 2.2 times less than that from the oil paste. Besides, beta-carotene absorbed from these oily drugs retained in the blood serum for longer period than that form Cyclocar.

[The cytochrome P-450-dependent mono-oxygenase system of the liver and interleukin-1 production by the macrophages in adjuvant arthritis in rats under the influence of beta-carotene]. / Tsitokhrom P-450-zavisimaia monooksigenaznaia sistema pecheni i produktsiia interleikina-1 makrofagami pri ad''iuvantnom artrite u krys pod vliianiem beta-karotina.

A relationship between the production of interleukin 1 (IL-1) by macrophages from adjuvant-induced arthritic rats and cytochrome P-450-dependent hepatic microsomal monooxygenase was studied. The synthesis of IL-1 by splenic and peritoneal macrophages on day 17 postadjuvant treatment was not altered, but the hepatic cytochrome P-450 levels and monooxygenase activity were significantly decreased. Beta-carotene treatment of arthritic rats reduced hind paw swelling and concurrently stimulated the ability of macrophages to secrete IL-1 and increased the cytochrome P-450 levels and the activity of hepatic monooxygenase. The findings did not establish a definite relationship between the production of IL-1 by systemic macrophages on the one hand, and the hepatic cytochrome P-450 levels a and monooxygenase activity on the other hand. It thus appears that IL1 is unable to play a role of a mediator between the immune system and the hepatic cytochrome P-450-dependent monooxygenase system of rats with adjuvant-induced arthritis.

[The effect of beta-carotene on the development of adjuvant arthritis and interleukin-1 production in rats]. / Vliianie beta-karotina na razvitie ad''iuvantnogo artrita i produktsiiu interleikina-1 u krys.

The production of interleukin 1 (IL1) by peritoneal and splenic macrophages from rats with adjuvant-induced arthritis on day 17 postadjuvant treatment was not altered compared with normal. Treatment of arthritic rats with beta-carotene reduced hind paw swelling and significantly increased ability of macrophages to secrete IL1 as well as stimulated spontaneous proliferation of splenic lymphocytes. No direct relationship between the release of IL1 from peritoneal and splenic macrophages and increase of hind paw swelling was revealed.

Kinetics of soybean lipoxygenase reaction in hydrated reversed micelles.

The rate of linoleic acid peroxidation catalysed by soybean lipoxygenase I was studied as a function of the hydration degree of aerosol OT (bis(2-ethylhexyl) sulfosuccinate sodium salt) reversed micelles in octane. Lipoxygenase reaction parameters for the micelle-bound substrate were spectrophotometrically determined. The linoleic acid distribution between the micelles and octane was detected by the sedimentation method, with the concentration of linoleic acid in supernatant after settling of micelles (i.e. the concentration of free linoleic acid) being estimated by the enzymatic method. The apparent constant of linoleic acid distribution (the ratio of the bound and free substrate concentrations) was enhanced with increasing hydration of reversed micelles. The dependence of the enzymatic reaction rate on the bound substrate concentration obeyed the empiric Hill equation. The Hill coefficient remained practically constant (h = 1.34) as the hydration degree changed. Parameters of the lipoxygenase reaction, enzyme reaction limiting rate V and semi-saturation substrate concentration [S]0.5 increased with increasing degree of hydration and reached the optimum at [H2O]/[AOT] approximately 30, where dimensions of the micellar internal cavity coincided with those of the enzyme molecule. Some aspects of kinetic behavior of membrane-bound enzymes participating in chemical transformation of non-polar compounds dispersed in lipid phase are discussed.

A novel approach to study of action of water-insoluble inhibitors of enzymic reactions.

The effect of water-insoluble compounds on enzyme catalytic properties was studied using a colloidal solution of water in organic solvent as reaction medium. In this microheterogeneous medium enzyme is entrapped into hydrated reversed micelles of a surfactant, the dimensions of the internal hole of the micelles being dependent on the ratio of water to surfactant. At sufficiently low values of this ratio the molecule of entrapped enzyme has limited mobility in the micelle. Because of this the interaction of the enzyme with water-insoluble compound which is added in assay solution and intercalated in the surface layer of the micelle may be manifested. The suggested method was used to study the inhibitory action of dihydroriboflavin esters on D-amino acid oxidase from pig kidney and soybean lipoxygenase. The reaction medium was hydrated reversed micelles of Aerosol OT in octane. The method of sedimentation in an analytical ultracentrifuge has shown the dihydroriboflavin esters to be completely included into reversed micelles.

[Changes in the lipolytic enzymatic activity of the blood in rats maintained on an atherogenic diet]. / Izmenenie aktivonosti lipoliticheskikh fermentov v plazme krovi krys pri soderzhanii ikh na aterogennom ratsione.

The paper discusses changes in the activity of lipolytic enzymes in blood plasma of rats before and after heparin administration in the course of experimental atherosclerosis development. This development was shown to be characterized by an abrupt rise of preheparin plasma level (comparatively to normal) and of monoacylglycerolipase and tributyrinase activity, with this rise being unchanged throughout the experiment. An increase in the postheparin plasma activity of heparin-dependent lipolytic enzymes, lipoprotein lipase, triacylglycerolipase, monoacylglycerolipase, and tributyrinase was seen only in the initial stage of the disease. After 30-40 weeks the activity of the enzymes was below normal. It is concluded that the increased activity of lipoproteid lipase and triacylglycerolipase is related to their activation in the blood channel.

[Determination of the activities of lipoprotein lipase, triacylglycerolipase, monoacylglycerolipase, and tributyrinase in the plasma, myocardium, and cardiac adipose tissue of rabbits]. / Opredelenie aktivnosti lipoproteidlipazy, triatsilglitserolipazy, monoatsilglitserolipazy i tributirinazy v plazme krovi, miokarde i zhirovoi tkani serdtsa krolikov.

Fatty emulsion intralipid, activated by rabbit blood plasma, intralipid, micellar emulsion of monooleine and tributyryne emulsion were used, respectively, as substrates for estimation of asctivities of lipoprotein lipase (EC 3.1.1.34), triacyl glycerolipase (EC 3.1.1.3), monoacyl glycerolipase (EC 3.1.1.23) and tributyrynase (EC 3.1.1.13) by modified Dol's method in rabbit heart tissue, heart fatty tissue and blood plasma. Effect of heparin on extraction of lipolytic enzymes from tissues was studied.

[Use of potentiometric titration at constant pH for determination of the activity of lipoprotein lipase]. / Primenenie metoda potentsiometricheskogo titrovaniia pri postoiannom pH dlia opredeleniia aktivnosti lipoproteidlipazy

A method is developed for determination of the lipoprotein lipase activity. The method is based on the steady state potentiometric titration at constant pH value of higher fatty acids, liberated during the hydrolysis. An oil emulsion intralipid, activated by human blood serum, was used as a substrate. The sensitivity of the method was equal to 0.004-0.010 micronM of fatty acids per min. The reproducibility of the results was 2-5%. This simple and rapid method enabled to study kinetic of reactions, catalyzed by lipoprotein lipases.