[Multilocus genotyping of polymorphous STR-loci of chromosomal DNA in individual cells: technical difficulties].

This study was designed to estimate the effectiveness of special technical procedures for the enhancement of sensitivity of multiplex analysis of DNA, such as the use of low-plexity PCR systems and the whole genome preamplification technology, and the possibility of their application for the purpose of forensic medical genotyping of polymorphous STR-loci of chromosomal DNA in individual cells. The authors refused to use the imitation model (equivalent DNA dilutions) for the sake of obtaining the maximally informative data and chose to work with real preparations of solitary buccal epithelial cells isolated by the laser microdissection technique. It was shown that neither the use of the low-plexity multilocus PCR systems nor the whole genome pre-amplification technology makes possible reliable genotyping of STR-loci of chromosomal DNA in individual cells. The proposed techniques allow for DNA genotyping in preparations consisting of 10 diploid cells whereas the methods for reliable genotyping of STR-loci of chromosomal DNA in individual cells remains to be developed.

[The functional tests in clinical diagnostic laboratory: the detection of magnesium deficiency in the loading test].

The article deals with the value and role of functional tests in practice of clinical diagnostic laboratories. The possibilities of evaluation of biological function of homeostasis according the changes of magnesium ions or calcium concentration in urine or blood hence reflecting the deficiency of these ions in vivo. The magnesium tolerant test is described It is demonstrated that it can be applied both in curative preventive institutions and ambulatories. In the examined group of patients, 78% had physiologic parameters of magnesium concentration, 17% suffered from hypermagnesiumuria and 5%--from hypermagnesiumuria. The magnesium deficiency of different degree was detected in 87% of patients. In the most part of patients with magnesium deficiency normomagnesiumuria was detected. Only in one case with normomagnesiumuria the magnesium deficiency was absent. In 30% of patients with magnesium deficiency the concentration of cation in day urine decreased up to 2.2 times after load dose. In absence of deficiency the monotony of cation's excretion was noted. Under the magnesium deficiency the character of process changed but velocity of excretion of magnesium after load probe slightly decreased relative to values before the load. The impact of alcohol under established magnesium deficiency results in increasing of velocity of excretion of this analyte. In patient with diabetes mellitus type II six months before the diagnosis of this disease the hidden deficiency of magnesium was detected. The magnesium deficiency was not detected after the antidiabetic treatment was applied The results permit to postulate the possibility of application oral load test with magnesium to assess the impact of various stress, physical, emotional and psychological factors. The detection of magnesium deficiency permit to broad the complex treatment, to accelerate and to enhance the results of treatment of diseases. Besides, the evaluation of patient's condition according the reaction of the organism to the probe with magnesium load is an essential element of personalized medicine.

[Determination of antibodies against cyclic citrullinated peptide in rheumatoid arthritis].

The ability of the synthetic peptide IMG-5, that reproduces one of the antigenic determinants of the protein filaggrin, to show antigenic activity was studied when anti-cyclic citrullinated peptide antibodies (ACCPAb) to filaggrin were found in the serum samples of patients with rheumatoid arthritis (RA). The binding of IMG-5 to ACCPAb has been shown to be specific (dose-dependent and reversible). The serum samples from patients with RA, controls, and donors show a significant difference in the interaction of the synthetic peptide with ACCPAb (p < 0.005 and p < 0.0001, respectively). The level of IgM rheumatoid factor (RF) detected in patients with RA differs greatly from that in the controls. In the patients with RA versus the controls, the specificity of ACCPAb determination was as high as 87; and the sensitivity was 40.5%. When ACCPAbs were determined using the commercial kit CCP, the specificity and sensitivity were 94 and 47.3%, respectively. The specificity of RF detection was equal to 50% and the sensitivity was 70%. The sensitivity of the test using IMG-5 is a maximum in X-ray stage IRA (69.2%) and falls in its stage III (26.7%). On the contrary, the sensitivity of the commercial kit and RF determination increases from X-ray stage I (46.2 and 53.8%, respectively) to II (66.7 and 80%). The sensitivity of the used tests in varying RA activities has demonstrated that they are most effective in patients with moderate RA activity. The concurrent detection of ACCPAb and RF increases the probability of differentiating RA from other rheumatic diseases.

[Serum nitrates in patients with systemic lupus erythematosus and antiphospholipid syndrome]. / Uroven' nitratov v syvorotke krovi patsientov s sistemnoi krasnoi volchankoi i antifosfolipidnym sindromom.

AIM: To elucidate clinical implications of nitrates (NO3) concentration as an indicator of nitric oxide (NO) level in blood serum of patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS). MATERIAL AND METHODS: Blood serum concentration of NO3 was measured by high-performance liquid chromatography in a total of 41 patients with SLE (n = 17), PAPS (n = 14) and secondary APS (n = 9). Sera from 10 healthy subjects (donors) served as control. NO3 concentration > 40 mcmol/l was stated high. SLE activity was assessed by V. A. Nasonova's scale and SLEDAI index (SLEDAI > 9 indicated SLE exacerbation). Patients with PAPS were divided into two groups depending on clinical symptoms: 7 patients with significant thrombosis or history of more than two thrombotic complications (group 1); 7 patients with the history of two or less thromboses (group 2). RESULTS: The mean serum nitrate concentration in the SLE group was 43.59 mcmol/l (range 17.06-113.22). The nitrate level of the sAPS + SLE group was 49.81 +/- 27.54 and did not significantly differ from APS (33.67 +/- 14.70). By univariate standard analyses, serum nitrate concentration was significantly higher in patients with high disease activity (57.31 +/- 25.12 vs. 25.62 +/- 6.96, Mann-Whitney test, p < 0.01). The Spearman correlation coefficient of nitrate level with SLEDAI was 0.8 (p < 0.001). CONCLUSION: A nitrate level is increased in patients with active SLE and in APS patients with severe thrombotic complication.

[Determination of nitric oxide in serum and plasma of human blood. Method of high pressure liquid chromatography]. / Opredelenie oksida azota v syvorotke i plazme krovi cheloveka. Metod vysokoéffektivnoi zhidkostnoi khromatografii.

Two methods of measurement of nitrite and nitrate determination in human serum and plasma (a colorimetric method based on Griess reaction and a HPLC method) have been compared HPLC method revealed extremely low level of nitrites blood plasma, which was roughly the same (about 3 microM or less) in healthy donors, patients with diabetes and hypertension. The level of nitrates in the group of healthy donors (29.9 +/- 2.8 microM) is lower than in patients with diabetes (58.6 +/- 6.9 microM), and in patients with hypertension (35.4 +/- 2.5 microM). Comparison of data obtained by colorimetric and HPLC methods revealed close correlation (r = 0.77, p < 0.001). Methodical requirements for preparation of samples of serum and plasma of blood for analysis and correct intepretation of results are discussed in view of biochemical properties of nitric oxide molecule.

[Effect of atorvastatin on endothelial function in patients with familial hypercholesterolemia]. / Vliianie atorvastatina na funktsional'noe sostoianie éndoteliia u bol'nykh s nasledstvennoi giperkholesterinemiei

AIM: To study the influence of treatment with HMG-CoA reductase inhibitor atorvastatin on endothelial function in patients with familial hypercholesterolemia type IIa. MATERIALS AND METHODS: Sixteen patients (5m/11w, 51-/+3 years) with familial hypercholesterolemia were studied before and after 3 months of therapy with atorvastatin 20 mg/day. EDRF release test (D.Celermajer, 1992) was used to assess flow-mediated endothelium-dependent vasodilatation (FMD) of the brachial artery in response to reactive hyperemia. Plasma nitrite/nitrate (NOx) levels were measured as an indirect index of nitric oxide (NO) production in vivo using HPLC. RESULTS: Atorvastatin treatment resulted in a 32% reduction in total serum cholesterol (CH), 41% reduction in low density lipoprotein (LDL) CH, 16% reduction in triglycerides and a 21% increase in high density lipoprotein CH. Flow mediated dilatation (FMD) was impaired at baseline (5.8-/+0.9%) and significantly improved up to 9.5-/+0.9% after 3 month atorvastatin therapy (p<0.002). Change in FMD inversely correlated with baseline FMD (r = -0.58, p<0.05). There was no significant correlation between FMD and neither total serum CH nor LDL CH levels at baseline. During atorvastatin therapy significant reduction of plasma NOx levels occurred from 53.4-/+5.1 mcmol/l at baseline (range 42.6-86.2 mcmol/l) to 35.5-/+5.1 mcmol/l (18.4-46.0 mcmol/l) after treatment (p<0.02, n=7). CONCLUSION: In patients with familial hypercholesterolemia atorvastatin produced beneficial effect on endothelial function (increase in flow-mediated dilatation, decrease in NOx).

[Cardiolipin antibodies dependents on beta2-glycoprotein-1 in antiphospholipid syndrome]. / Zavisimye ot beta2-glikoproteina-1 antitela k kardiolipinu pri antifosfolipidnom sindrome.

AIM: To study beta2-GP-I-dependent binding of phospholipid antibodies (PAb) to phospholipids and this process participation in pathogenesis of antiphospholipid syndrome (APS). MATERIALS AND METHODS: IgG-fractions and sera from 20 patients with APS. Cofactor activity of beta2-GP-I isolated from serum of healthy donors was examined with modified immunoassay. RESULTS: Contrary to donor IgG, binding of IgG fractions isolated from sera of APS patients with cardiolipin grows dose-dependently in the presence of beta2-GP-I. Cofactor activity of beta2-GP-I is confirmed in the study of sera of APS patients. Sera containing beta2-GP-I-dependent antibodies to cardiolipin (aCL), unlike aCL-negative sera, react with solid-phase immobilized beta2-GP-I. CONCLUSION: It is confirmed that beta2-GP-I participates in interaction of PAb with cardiolipin. Pathogenetic implication of beta2-GP-I-dependent PAb for onset of APS is discussed.

[Synthesis and biological properties of artificial antigens based on 280-289 fragment of beta2-gpI]. / Sintez i biologicheskie svoistva iskusstvennykh antigenov na osnove peptida, sootvetstvuiushchego posledovatel'nosti 280-289 belka beta2-gpI.

A series of artificial antigens were synthesized on the basis of the FC(Acm)KNKEKKC(Acm)S peptide from the beta 2-glycoprotein I sequence: lipophilic analogues, the peptide-BSA conjugate, and multiple antigen peptide (MAP) containing eight copies of the peptide on an oligolysyl core. The solid phase method for acylation of the peptide with fatty acids and the HPLC analysis of the acylpeptides were described. Antigenic properties of the resulting compounds were evaluated by CL-ELISA.

[Synthesis and antigenic properties of peptide fragments of beta2-glycoprotein-I]. / Sintez i issledovanie antigennykh svoistv peptidnykh fragmentov beta2-glikoproteina-I.

A number of beta 2-glycoprotein-I peptide fragments were synthesized by using the Fmoc-scheme of the solid phase method. Antigenic properties of these peptides were determined by ELISA. Acm-protected FCKNKEKKCS peptide was shown to inhibit binding of anti-cardiolipin antibodies to cardiolipin.

[Determination of free and bound phenol in urine during mass screening of the populace]. / Opredelenie svobodnogo i sviazannogo fenola v moche pri massovom obsledovanii naseleniia.

Quantitative estimation of phenol content in human urine involving Gibbs reagent was described in detail; the procedure was applied for screening test of people. The Gibbs reagent was synthesized from commonly available substances 2,6-dibromine-4-aminophenol and 4-nitrophenol. Estimation of total and bound forms of phenol was carried out in 0.5 ml of urine, while phenol conjugates were hydrolyzed by means of heating of urine samples containing 0.2 ml of 42% perchloric acid in boiling water bath and the phenol released was extracted by 3.0 ml of diethyl ether. Sensitivity of the procedure was about 0.1 micrograms of phenol in a sample, when microphotometer, applied in immunological studies, was used for measurement of optic density in the plates holes. Content of total phenol was estimated in urine of 15 children of 6-6.5 years old from Moscow using the procedure developed and it constitute from 2.5 micrograms/ml to 47.0 micrograms/ml, middle value was 9.5 micrograms/ml. In 47 children from Noyabrsk town these parameters constituted 1.5 micrograms/ml, 150 micrograms/ml and 19 micrograms/ml, respectively in adults - 1.5 micrograms/ml, 168 micrograms/ml and 167.7 micrograms/ml, respectively. The upper limit of the phenol normal concentration in urine was proposed to be as 50 micrograms/ml in screening test of people. Content of phenol in urine should be measured within a day during individual examination.

[Antibodies to pathogens of Lyme's disease in patients with myocarditis and dilated cardiomyopathy]. / Antitela k vozbuditeliu bolezni Laima u bolnykh miokarditom i dilatatsionnoi kardiomiopatiei.

The paper presents the information on the involvement of the cardiovascular system in the patients with Lyme's disease as well as the results of serological investigation in patients with myocarditis and dilatation cardiomyopathy for the presence of antibodies to the causative agent of Lyme's disease with the aid of enzyme immunoassay. Out of 71 sera 3 specimens had the values of optical density higher than the upper normal limits. Based on the investigation conducted and the analysis of the clinical picture, the authors suspected a Borrelia origin of these cardiac involvements. However, to verify the diagnosis morphological examination of endomyocardiac biopsy specimens was found to be mandatory.

[Cryoglobulinemia and fibronectin in systemic lupus erythematosus]. / Krioglobulinemiia i fibronektin pri sistemnoi krasnoi volchanke.

EIA was used to measure the concentration of fibronectin in plasma and cryoprecipitates of 37 patients suffering from systemic lupus erythematosus coupled with cryoglobulinemia. A definite relationship was discovered between the level of cryoglobulins and the activity of SLE, the concentration of fibronectin in plasma and liver damage. A tendency was revealed towards increase of the fibronectin content in plasma of patients with a high level of antibodies to native DNA and CIC as was a significant correlation between the concentration of cryoglobulin protein and the concentration of fibronectin in cryoprecipitates.

[Use of solid-phase Clq-ELISA for detecting circulating immune complexes in human diseases]. / Ispol'zovanie tverdofaznogo Clq-immunofermentnogo metoda dlia opredeleniia tsirkuliruiushchikh immunnykh komplksov pri zabolevaniiakh cheloveka.

The authors provide the results of studying the methodologic characteristics and of the clinical trial of the C1q-ELISA for detection of the circulating immune complexes. The C1q-ELISA is shown to have a high sensitivity and to make it possible to identify 3 micrograms/ml and more of aggregated gamma-globulin. The method can use for detection of CIC E (ab')2-fragments of antibodies to IgG of man and protein A labeled with peroxidase. Using the method, the circulating immune complexes were identified in the sera of 50% of patients with systemic lupus erythematosus, of 30% of patients with dilated cardiomyopathy and of 53% of patients with nonspecific aortoarteritis. It is concluded that the C1q-ELISA can be used in clinical practice for detecting the circulating immune complexes in different diseases of man.

[Lateralization of opioid receptors and their putative ligands in the visual cortex of the turtle]. / Lateralizatsiia opioidnykh retseptorov i ikh predpolagaemykh ligandov v zritel'noi kore cherepakhi.

Opioid mu-agonist morphine, delta-agonist D-Ala2,D-Leu5-enkephalin (DADL) and kappa-agonist bremazocine locally applied to the surface of turtle visual cortex inhibited the orthodromic evoked potential (EP; fast negative component N1). The lack of cross-desensitization to the inhibitory action of opioids upon EP indicates that the drugs exert their effects via different opioid receptors. Morphine and bremazocine predominantly inhibited the left cortex EP, whereas DADL was a potent inhibitor of the right cortex EP. Thus opioid receptors which modulate evoked electrical activity of the left visual cortex (LVC) apparently belong mostly to mu- and kappa-type while delta-receptors were predominantly responsible for the modulation of electrical activity in the right visual cortex (RVC). Application of LVC- and RVC-extracts to the cortex surface led to EP inhibition, which was partially (60-80%) prevented by antagonist naloxone. LVC-extract proved to be a more potent inhibitor of the left cortex EP, whereas RVC-extract was found to be more effective when applied to the right cortex. It is suggested that not only opioid receptors, but also their endogenous ligands are lateralized in turtle visual cortex.

[Ability of the opiate kappa-agonist bremazocine to facilitate the flexor reflex of the right hindlimb in spinal rats]. / Sposobnost' opiatnogo kappa-agonista bremazotsina oblegchat' fleksornyi refleks pravoi zadnei lapy u spinal'nykh krys.

It has been found that bremazocine predominantly enhances the flexor reflex of the right hind limb, without affecting the left limb. The similarity of the visual data measuring the asymmetry of the pose and the asymmetry of EMG responses to painful stimulation has a high probability. No correlation between the asymmetry of the pose and EMG extensor activity has been found.

[Opioids evoke postural asymmetry in rats: the side of the flexed paw depends on the type of opioid agonist]. / Opioidy vyzyvaiut asimmetriiu pozy u krys : storona fleksirovannoi lapy zavisit ot tipa opioidnogo agonista.

Opioid kappa-agonists bremazocine and dynorphin (1-13), sigma-agonist SKF 10.047 and delta-agonist D-Ala2, D-Leu5-enkephalin (DADL) induce postural asymmetry of rats hind limbs under subarachnoidal administration below the level of spinal cord section (T3-T4). The side of the flexed leg depends on the opioid agonist type: bremazocine and dynorphin (1-13) induce predominantly right flexion. SKF 10.047--the left flexion, but not in all doses, DADL--in small doses (1 and 100 pg per animal)--of the right one, in larger doses (up to 10 ng per animal)--of the left one. Saline and opiate mu-agonist morphine do not induce postural asymmetry. Opiate antagonist naloxone prevents asymmetry development when injected prior opioid agonists, and also decreases the number of asymmetries induced by these agonists. Naloxone alone does not influence the per cent of animals with pose asymmetry. The opioid receptors are involved in asymmetry development. The revealed ability of opioid kappa-, delta- and sigma-agonists may be based on lateralization of opioid receptors in the rat spinal cord.

[Characteristics of the development of postural asymmetry in rats as affected by the opioids met-enkephalin and the kappa-agonist bremazocine]. / Osobennosti razvitiia asimetrii pozy u krys pod deistviem opioidov met-énkefalina i kappa-agonista bremazotsina.

It has been found that bremazocine and met-enkephalin induce postural asymmetry in spinal rats under subarachnoidal and intravenous administration. Intravenous administration of bremazocine to intact animals--even if after it (an hour later) their spinal cord is sectioned--produces no asymmetry, i. e. the spinal cord section is necessary for asymmetry development. The magnitude of postural asymmetry and the side of limb flexion are not constant for each animal, but they change in time. Though, on the average, the percent of asymmetric animals and the ratio of left and right flexions in each group of animals are practically constant. When the spinal cord is sectioned at the T1-T4 level, the bremazocine and metenkephalin induce mainly the right-leg flexion: when the section is made at the T5-T9 level, the left-leg is bent, i. e. the flexion side depends on the level of the section. It is suggested that the ability of opioids to induce postural asymmetry is based on lateralization of opioid receptors in the rat spinal cord.