Antisera Against the Indolealkylamines: Tryptophan, 5-Hydroxytryptophan, 5-Hydroxytryptamine, 5-Methoxytryptophan, and 5-Methoxytryptamine Tested by an Enzyme-Linked Immunosorbent Assay Method

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.     

Dopamine antibodies in the pathogenesis of parkinsonism]

L-DOPA and dopamine (DA) binding antibodies were found in the blood serum of Parkinsonian patients and middle-aged and elderly normal persons. DA-binding serum gamma-globulins of parkinsonian patients injected into rat caudate nuclei induced the pathogenetic mechanism of Parkinson's syndrome (generator of pathologically enhanced excitation) in these brain part and evoked main parkinsonian symptoms (oligokinesia, rigidity, tremor). The serum gamma-globulins of Parkinsonian patients without Da-antibodies caused less pronounced EEG disturbances. Parkinsonian symptoms developed rarely and were shorter and less pronounced compared with the DA-antibody effect. The DA binding antibodies role in Parkinson's syndrome pathogenesis and is L-DOPA therapeutic tolerance formation was discussed.     

Monoclonal Anti-Conjugated Acetylcholine Antibody and Immunohistochemical Applications in Rat Nervous System

Acetylcholine (ACh) conjugates were injected into AKR and DBA mice over a period of 10 weeks. The polyclonal antisera were tested at various immunization times for affinity and specificity using an enzyme-linked immunosorbent assay (ELISA). The most immunoreactive compound was found to be choline-glutaryl-bovine serum albumin (or conjugated ACh). The AKR and DBA mice yielding the highest apparent affinity were killed, and the spleen cells were fused with X63 or SP2/O/Ag mouse myeloma cells. Supernatants of confluent cultures were tested for the presence of anti-conjugated ACh antibodies using the same ELISA method. The best results were obtained with the hybridomas from AKR spleen cells and X63 mouse myeloma cells. Monoclonal antibody affinity and specificity were then evaluated by a radioimmunological procedure using iodinated monoclonal anti-conjugated ACh antibody. From competition experiments, the most immunoreactive compound was choline-glutaryl-protein. The other related compounds were recognized either poorly or not at all. The high affinity and specificity of our monoclonal antibody enabled us to visualize ACh molecules on fixed rat brain sections. ACh was fixed with a mixture of nitrobenzyl alcohol and glutaraldehyde. Many ACh-immunoreactive cell bodies and fibers were seen on sections from the basal forebrain and spinal cord. Preadsorption and other immunohistochemical tests demonstrated that the ACh staining was highly specific.     

Actions of dopamine and related amines on reserpinized and chronically denervated vasa deferentia of the rat

1. Reserpine, chronic guanethidine denervation and alpha-adrenoceptor antagonists were used to distinguish between presynaptic and postsynaptic actions of exogenous dopamine (DA), octopamine (OA), tyramine (TA) and noradrenaline (NA) on the rat vas deferens. 2. TA has only a presynaptic action while DA and OA have mixed presynaptic actions (releasing endogenous NA) and postsynaptic actions. 3. The postsynaptic actions of DA and OA are likely to be mediated by alpha-adrenoceptors rather than by specific receptors.     

Effects of L-dopa and L-tyrosine on release of free and conjugated dopamine, homovanillic acid and dihydroxyphenylacetic acid from slices of rat striatum

Conjugation (presumably with sulfate) is a demonstrable metabolic pathway for 3, 4-dihydroxyphenylethylamine (dopamine, DA) in brain. Studies were done to determine whether conjugation becomes of increased significance in the presence of precursors of DA. The effects of 3, 4-dihydroxyphenylalanine (L-DOPA) and L-tyrosine on the efflux of free and conjugated DA, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid from slices from striatum in rats were studied under quiescent conditions and during release evoked by 40 mM K+ or by 5 X 10(-5) M phenylethylamine (PEA). Conjugated DA was present in the basal efflux from striatal slices and the amounts present were increased during evoked release. More conjugated DA was present in superfusate during K+-evoked release than during PEA-evoked release. L-Tyrosine (5 X 10(-4) M or 5 X 10(-5) M) had little effect on the efflux of conjugated DA, but decreased the amounts of free DA released by PEA, and attenuated the increase in DOPAC that occurred during K+-evoked release of transmitter. L-DOPA (5 X 10(-5) M) increased the formation of conjugated DA, but to a lesser extent than that of free DA or of DOPAC. Thus even after the addition of precursors, conjugation remains a minor metabolic pathway for DA relative to O-methylation or oxidative deamination. The data also suggest that conjugation of DA occurs chiefly outside of the dopaminergic neurons in striatum.     

Enzymatic alteration of C1q, the collagen-like subcomponent of the first component of complement, leads to cross-reactivity with type II collagen

Native serum C1q, the collagenous-like subcomponent of the first component of complement, is not recognized by polyclonal anti-collagen type II antibodies. However, when purified C1q was subjected to limited proteolysis by collagenase it showed antigenic cross-reactivity with collagen type II. The same cross-reactivity was observed with hemolytically active C1q in synovial fluids of patients with rheumatoid arthritis (RA), whereas C1q from synovial fluids of patients with osteoarthritis (OA), villo-nodular synovitis and ankylosing spondylitis was not recognized by this antibody. However, incubation of synovial fluid C1q of OA patients with synovial fluid leucocytes from RA patients led to an alteration of OA-C1q which was now recognized by the anti-collagen type II antibody.     

The effect of active immunization with a conjugate of dopamine and bovine serum albumin on the development of an experimental MPTP-induced depressive syndrome and on the monoamine metabolism in the brain of rats

Active immunization with dopamine conjugated with bovine serum albumin (DA-BSA) or BSA with complete Freund's adjuvant (CFA) partly suppressed the development of the MPTP-induced depressive syndrome in rats preventing the appearance of "behavioral despair" symptoms: increase in immobility time and higher index of depression in forced-swim test. In DA-BSA-immunized rats the content of DOPA, DA, HVA, NA, and 5-HN in caudate putamen and that of NA in the frontal cortex was increased, while in BSA-immunized rats the content of 5-HT in both brain areas and that of DOPAC in the frontal cortex was decreased both in rats with reduced depressive syndrome and in saline control as compared with intact animals a day after the last drug injection. In DA-BSA-immunized rats with reduced depressive syndrome the increase in DA and 5-HT content in caudate putamen was less expressed and DOPAC content was lower than in saline control. In BSA-immunized depressive rats DA content in the frontal cortex was also reduced as compared to control.     

Factors involved in the production of specific antibodies to estriol and estradiol

To determine what effect the site of attachment of the hapten would have on specificity, 6-oxoestrogens were synthesized and conjugated to several carrier proteins, using the mixed anhydride formed between isobutyl chloroformate and the steroid 6-oxime. Estriol was also treated with phosgene to produce a mixture of chloroformates followed by conjugation to bovine serum albumin. Rabbits were immunized, and the specificity of the anti-sera produced was established by cross-reaction measurements, using both direct binding and binding inhibitor assays. Comparison of the immune response showed that antibodies to the randomly linked estriol chloroformate had a high degree of cross-reactivity with estrone and estradiol (30–100%), while those produced to 6-oxo linked estriol showed only a 1–12% cross-reaction. Similar results were obtained for estradiol antibodies. Comparison of antibody formation and specificity of 6-oxoestriol linked to a polylysine copolymer, bovine serum albumin or rabbit serum albumin was made. Also, the antigen was given with complete Freund's adjuvant, adsorbed onto charcoal, entrapped within polyaery lamide gel or in vivo titrated with estrogen implants. Only estrogen specifically linked to bovine serum albumin in Freund's adjuvant gave satisfactory results, while the others yielded antibodies of decreasing titers or low specificity.It is concluded that specificity and titers seem to depend not only on the site of conjugation but also on the carrier, immunization procedure and certain other factors.     

Regulation of pyridoxal-5′-phosphate level by biogenic amines in mouse brain

We investigated the relationship between the concentration of pyridoxal-5′-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.     

Human prostaglandin H synthase (hPHS)-1- and hPHS-2-dependent bioactivation, oxidative macromolecular damage, and cytotoxicity of dopamine, its precursor, and its metabolites

The dopamine (DA) precursor l-dihydroxyphenylalanine (L-DOPA) and metabolites dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine may serve as substrates for prostaglandin H synthase (PHS)-catalyzed bioactivation to free radical intermediates. We used CHO-K1 cells expressing human (h) PHS-1 or hPHS-2 to investigate hPHS isozyme-dependent oxidative damage and cytotoxicity. hPHS-1- and hPHS-2-expressing cells incubated with DA, L-DOPA, DOPAC, or HVA exhibited increased cytotoxicity compared to untransfected cells, and cytotoxicity was increased further by exogenous arachidonic acid (AA), which increased hPHS activity. Preincubation with catalase, which detoxifies reactive oxygen species, or acetylsalicylic acid, an inhibitor of hPHS-1 and -2, reduced the cytotoxicity caused by DA, L-DOPA, DOPAC, and HVA in hPHS-1 and -2 cells both with and without AA. Protein oxidation was increased in hPHS-1 and -2 cells exposed to DA or L-DOPA and further increased by AA addition. DNA oxidation was enhanced earlier and at lower substrate concentrations than protein oxidation in both hPHS-1 and -2 cells by DA, L-DOPA, DOPAC, and HVA and further enhanced by AA addition. hPHS-2 cells seemed more susceptible than hPHS-1 cells, whereas untransfected CHO-K1 cells were less susceptible. Thus, isozyme-specific, hPHS-dependent oxidative damage and cytotoxicity caused by neurotransmitters, their precursors, and their metabolites may contribute to neurodegeneration associated with aging.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

Studies on the mechanism of post-etorphine catalepsy. Modyfying effect of amphetamine, apomorphine and dihydroxy-phenylalanine (L-dopa) on etorphine-induced concentrations of dopamine and noradrenaline in the rat central nervous system

Studies on the mechanism of post-etorphine catalepsy. Modyfying of amphetamine, apomorphine and dihydroxyphenyl-alanine (L-DOPA) on etorphine-induced concentrations of dopamine and noradrenaline in the rat central nervous system. Acta Physiol. Pol., 1979, 30 (2): 279--287. During stereotypy induced with amphetamine, apomorphine and 1-dihydroxyphenylalanine (L-DOPA) increased concentrations of dopamine (DA) and noradrenaline (NA) were found in the motor centres of the central nervous system (CNS). In post-etorphine catalepsy the concentrations of DA and NA were also increased in the frontal cortex, striopallidum, pons and cerebellum and in the lumbosacral spinal cord. However, these stereotypy-inducing agents used in premedication of post-etorphine catalepsy delayed significantly its onset and reduced its duration.     

ACCUMULATION AND DISAPPEARANCE OF NORADRENALINE AND ITS MAJOR METABOLITES SYNTHESIZED FROM INTRAVENTRICULARLY INJECTED [3H]DOPAMINE IN THE RAT BRAIN

Abstract— A new combined ion-exchange and thin-layer-chromatographic procedure is described which separates and measures quantitatively, after intraventricular injection of [³H]dopamine (DA), the rat brain content of labelled noradrenaline (NA) and the following labelled noradrenaline metabolites: free 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), conjugated MOPEG, free plus conjugated dihydroxyphenylethyleneglycol (DOPEG), vanillic mandelic acid (VMA) and normetanephrine (NM). Labelled dopamine and its metabolites were also measured. The time-course study performed from 5 min to 24 h after [³H]DA showed that MOPEG and DOPEG, mainly as conjugates, are major NA metabolites whereas VMA is a very insignificant NA metabolite in the rat brain. A very rapid initial increase of [³H]NM, free MOPEG and conjugated MOPEG was found during the time interval where the [³H]NA biosynthesis is very high (0–15 min). This combined with the finding that these metabolites stabilize at lower levels during the [³H]NA ‘storage phase’ (9–24 h) provides a strong indication that newly synthesized NA preferentially is metabolized. Our measurements of endogenous NA, free MOPEG and conjugated MOPEG provide additional support. The injections of various decreasing doses of [³H]DA (3·08–0·0010 μg) showed that the proportions of total [³H]MOPEG and total [³H]DOPEG to [³H]NA were constant after all [³H]DA doses investigated. This finding indicates that the [³H]NA synthesized in situ behaves as a tracer, even after injections of non-tracer doses of [³H]DA. The results seem thus to indicate that the present technique provides a powerful tool for the investigations on central noradrenaline metabolism.     

Ascorbic acid suppresses the deconjugation of noradrenaline but not dopamine in plasma

The characteristics of hydrolysis of sulfoconjugated noradrenaline (NA) and dopamine (DA) in plasma using sulfatase were investigated. Ascorbic acid has been used as an antioxidant during the hydrolysis of conjugated NA or DA. Hydrolysis of NA sulfates was considerably inhibited by adding ascorbic acid (0.5-10 mM), and slightly inhibited by adding dithiothreitol (1-10 mM). In contrast, the hydrolysis of DA sulfates was not affected after either ascorbic acid or DTT treatment. On the basis of these findings, the levels of NA sulfates previously reported are found to be markedly lower than the actual levels of NA sulfates in human plasma.     

Selective antibodies to methadone enantiomers: synthesis of (R)- and (R,S)-methadone conjugates and determination by an immunoenzymatic method in human serum

Selective antibodies to (R)-methadone (Mtd) and to its racemate were produced in rabbits by immunization with conjugates of (R)- or (R,S)-hemisuccinyl-methadol-bovine serum albumin, respectively. A hapten was first prepared by reduction of (R)- or (R,S)-Mtd with sodium borohydride, followed by esterification with succinic anhydride. The conjugation of hapten with albumin was achieved by the mixed anhydride method. After immunization of rabbits, the titers and specificity of each antibody were determined by ELISA. The antibodies obtained were tested with (R)-, (S)-, (R,S)-Mtd, its major metabolite (EDDP), and some drugs of abuse (morphine, codeine, cocaine). The sensitivities of antibodies to (R)- and (R,S)-Mtd were about 1 and 2 ng/ml, respectively. Selective (R)-antibodies recognized (R)-Mtd about 40 times more avidly than the (S)-isomer, while an antiserum against (R,S)-Mtd recognized (R)- and (S)-isomers to about the same degree. Both selective antibodies showed little interference (about 0.5%) with EDDP metabolite and no crossreactivity with morphine, codeine, and cocaine. These two selective antibodies were used to develop an immunoenzymatic method (ELISA) for the determination of (R)- and (R,S)-Mtd in serum samples of patients under maintenance treatment for narcotic addiction.     

Investigation of Dopamine Content, Synthesis, and Release in the Rabbit Retina In Vitro: I. Effects of Dopamine Precursors, Reserpine, Amphetamine, and l-DOPA Decarboxylase and Monoamine Oxidase Inhibitors

The basal catecholamine content of rabbit retina was determined by liquid chromatography with electrochemical detection (LC-EC) and 3,4-dihydroxyphenylethylamine (dopamine, DA) found to be the major catecholamine. The immediate DA precursor, 3,4-dihydroxyphenylalanine (L-DOPA), and the metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were also detected at about 2.8% and 17% of DA levels, respectively. When added exogenously, L-tyrosine did not increase the rate of DA synthesis over the basal level. In contrast, exogenous L-DOPA led to a 3.5-fold increase in DA, and to a 20-fold increase in DOPAC content. The monoamine oxidase inhibitors pargyline and (-)-deprenyl differentially affected the degradation of DA, since 100 microM pargyline was apparently more effective than 100 microM (-)-deprenyl. Reserpine and (+/-)-amphetamine each induced a Ca2+-independent decrease of DA stores. The separate actions of reserpine and (+/-)-amphetamine in lowering tissue DA levels were additive, suggesting two separate pools of DA available for release from presynaptic stores. The present study demonstrates that the LC-EC technique may be used to investigate the modulation of the synthesis and release of retinal DA in vitro, without the prior uptake of radiolabelled transmitter.     

Selective antibodies to propranolol enantiomers produced from a new conjugate

A selective antibody to (S)-propranolol enantiomer was produced in rabbits by immunization with a new conjugate of N-aminopropylpropranolol-albumin. A hapten was first prepared by condensing (S)-propranolol or the racemate with 3-bromopropylphthalimide followed by hydrazinolysis, and the resulting compound conjugated to serum albumin by means of a glutaraldehyde- or carbodiimide-mediated reaction. Rabbits were immunized, and titres and specificity of antibodies were determined by ELISA. The antibodies obtained were tested with (S)-, (R)-, (R, S)-propranolol, and other structural analogs. Selective (S)-antibodies recognized (S)-propranolol 20 times more avidly than (R)-isomer while an antiserum against (R, S)-propranolol recognized both (S)- and (R)-isomers to about the same degree. ©1993 Wiley-Liss, Inc.     

A method for very rapid determinations of catechols using ion-pairing reverse phase HPLC with electrochemical detection: effects of L-dopa treatment on the catechol content in various rat brain structures

A simple and rapid method for determination of 12 catechols (9 endogenous and 3 internal standards, i.s.) using ion-pairing reverse phase HPLC with electrochemical detection is presented. This study basically concentrates on the importance of optimizing the mobile phase composition in isocratic systems where ordinary 25 cm X 4.6 mm i.d. columns are used. Mobile phase compositions for three different purposes are reported: 1) separation of 9 endogenous catechols, possibly occurring in the samples, and 3 i.s. in a moderately short retention time (tR) (L-DOPA, DOPEG, alpha-Methyldopa (alpha-MeDOPA, i.s.), Noradrenaline (NA), DOPAC, Adrenaline (A), Dihydroxybensylamine (DHBA, i.s.), Norsalsolinol (NS), Dopamine (DA), Epinine (EPI), Salsolinol (S) and Isoprenaline (ISO, i.s.) within 11 min), 2) ultra rapid separation of detectable endogenous catechols except L-DOPA (NA, DOPAC, A, DHBA (i.s.), NS and DA within 5.2 min and with S within 5.7 min) and 3) moderately fast separation of detectable endogenous catechols (L-DOPA, NA, A, DHBA (i.s.), NS, DOPAC and DA within 7.6 min and with S within 10 min). By balancing the pH, concentration of organic modifier (2-propanol) and pairing ion (1-heptanesulphonic acid) as well as preconditioning new columns with more packing material (Nucleosil 5 micron C18) and to high pressures (5000 psi) for 7 days, very fast separations with good baseline resolution between the peaks are possible. The method was applied on L-DOPA treated rats (100 mg/kg), where the catechol content was analysed in 7 different brain structures during the time course of synthesis and degradation (4 hours) of catechols from L-DOPA.     

The stimulatory roles of catecholamines and acetylcholine in the regulation of gonadotropin release in ovariectomized estrogen-primed rats.

Injections of 2 mg of progesterone into ovariectomized estrogen-primed rats significantly increased serum LH and FSH concentrations 3, 5 and 8 hr later. Receptor blockers of noradrenaline (NA), dopamine (DA) or acetylcholine (ACH), phenoxybenzamine (20 mg/kg body weight), pimozide (1mg/kg body weight) or atropine (700 mg/kg body weight), respectively, prevented the progesterone-induced gonadotropin release. On the other hand, none of them blocked the gonadotropin release following unilateral electrochemical stimulation (100 microA for 60 sec) of the medial preoptic area which occurred 0.5 and 1.5 hr later, although pimozide or atropine reduced serum LH concentrations at 4.0 hr after stimulation. Furthermore, the sites of action of NA, DA and ACH with respect to LH release were examined by intracerebral implantation in ovariectomized estrogen-primed rats DA or ACH, when implanted unilaterally into the medial preoptic urea, induced a significant increase in serum LH 5 hr later, whereas NA decreased LH levels. Implantations of NA or ACH into the bed nucleus of the stria terminalis or the medial amygdala increased serum LH although the effect of NA into the latter was not statistically significant. Only implantations of NA among the three substances into the lateral septum induced LH release. These results suggest that all of NA, DA and ACH play stimulatory roles in the regulation of gonadotropin secretion, and that there are regional differences of their effectivenesses in releasing LH within the limbic-preoptic area.     

Effects of dopamine on spontaneous activity generated by isolated spinal cord in 16- to 20-day-old chick embryos

The effects were investigated of applying L-DOPA, dopamine (DA), and noradrenaline (NA) on spontaneous activity (cyclic fluctuations in electrotonic dorsal and ventral root (DR and VR) potentials generated by a section of spinal cord isolated from 16 to 20-day-old chick embryos. A low concentration of L-DOPA (30–150 µm) intensified operation of the spinal generator, giving rise to above-threshold rhythm (i.e., spike activity in the DR and the VR). At a high concentration, L-DOPA produced inhibition of generator operation, although spontaneous activity did intensify during subsequent washout of the substance, with the onset of above-threshold rhythm. Both DA and NA failed to affect spontaneous activity in the VR and the DR at a concentration to 50 µM but a concentration of 100 µM produced inhibition. Application of 20 µM 2-amino-5-phosphonovaleric acid blocked the reinforced spontaneous activity produced by low L-DOPA concentrations. Activity generated by the neuronal network of the isolated dorsal horn rose under the effects of low L-DOPA concentrations; rhythmic activity was observed neither before nor after applying this substance in isolated ventral horn. Findings obtained would point to the occurrence of a direct (i.e., non-catecholamine dependent) excitatory influence of L-DOPA on the neuronal network of the chick embryo dorsal horn.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 338–343, May–June, 1991.