Streptomyces tsukubensis VKM Aс-2618D-an Effective Producer of Tacrolimus.

The Streptomyces sp. VKM Ac-2618D strain has been identified, and its morphological and physiological features have been studied in relation to the production of the immunosuppressant tacrolimus. The phenotypic variability of the strain was analyzed, and a dissociant with a high level of tacrolimus production was selected. Based on a comprehensive study of morphological, physiological, and chemotaxonomic properties and on phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis are shown. The results broaden the understanding of the characteristics of polyketide biosynthesis and can be used in the development of technology for tacrolimus production.

Microbiological synthesis of stereoisomeric 7(α/ß)-hydroxytestololactones and 7(α/ß)-hydroxytestolactones.

Microbiological synthesis of 7α- and 7ß-hydroxy derivatives of testololactone and testolactone was developed based on bioconversion of dehydroepiandrosterone (DHEA) by fungus of Isaria fumosorosea VKM F-881 with subsequent modification of the obtained stereoisomers by actinobacteria. The first stage included obtaining of the stereoisomers of 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones in the preparative amounts. Then the conversion of 7-hydroxylated D-lactones obtained by selected actinobacteria of Nocardioides simplex VKM Ac-2033D, Saccharopolyspora hirsuta VKM Ac-666, and Streptomyces parvulus MTOC Ac-21v was studied. Under the transformation of 3ß,7α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one and its corresponding 7ß-stereoisomer by N. simplex VKM Ac-2033D and S. hirsuta VKM Ac-666 the 7α- and 7ß-hydroxy-17a-oxa-D-homo-androst-4-ene-3,17-dione (7α- and 7ß-hydroxytestololactone), 7α- and 7ß-hydroxy-17a-oxa-D-homo-androsta-1,4-diene-3,17-dione (7α- and 7ß-hydroxytestolactone) were obtained with molar yields in a range of 60.3-90.9 mol%. The crystalline products of 7α-hydroxytestololactone, 7α-hydroxytestolactone, and their corresponding 7ß-hydroxy stereoisomers were isolated, and their structures were confirmed by mass spectrometry and 1H-NMR spectroscopy analyses. The strain of Str. parvulus MTOC Ac-21v transformed 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones into the corresponding 3-keto-4-ene analogs and did not show 3-ketosteroid 1(2)-dehydrogenase activity. The activity of actinobacteria towards steroid D-lactones was hitherto unreported.The results contribute to the knowledge of metabolic versatility of actinobacteria capable of transforming steroid substrates and may be applied in the synthesis of potential aromatase inhibitors.

[Use of somatostatin analogues in the pancreonecrosis therapy (kliniko-experimental research)]. / Osobennosti primeneniya sinteticheskogo analoga somatostatina v terapii pankreonekroza (kliniko-eksperimental'noe issledovanie).

The article is devoted to the study and optimization of therapeutic strategy in pancreatic necrosis. We studied the effectiveness of clinical application of human somatostatin analog - octreotide in different dosages. Submitted investigationconsists of experimental and clinical parts. MATERIAL AND METHODS: Experimentalmodel of acute destructive pancreatitis onrats of Wistar line with weight of 300±30 gwas reproduced.There were 5 animal groups (one control group and four experimental groups). Experimental animals received oktreotidin different doses. In 2 days there were estimated the blood biochemistryand histopathologic features of liver and pancreas of operated animals. In clinical part there were formed two groups of patients out of selected case histories by a principle of similarity of complex therapy differing in oktreotid doses, namely: 1) pancreatonecrosis patients estimated of 3 to 8 points by Ranson scale in whose treatment regimen oktreotid 300 mkg/day (n=70) dose was included; 2) pancreatonecrosispatients estimated of 3 to 8 points by Ransonin whose treatment regimenoktreoid1200 mkg/day (n=38) was included. There were compared indices of lethal outcomes, average bed-days, and dynamics of laboratory findings, number and volume of surgical procedures. RESULTS AND CONCLUSION: Dose-related effect of a synthetic analogue of somatostatin, acetateoktreotid, was proved. Introduction in complex therapy of pancreatonecrosisthe highest possible authorized doses of oktreotid (1200 mkg/day) is conducive to more favorable course of illness, dynamic reduction of amilazemia, decrease of lethal outcomes and total bed-day, and also, reduction of initial and repeated operative procedures.

[11beta-Hydroxylation of 6alpha-Fluoro-16alpha-Methyl-Deoxycorticosterone 21-Acetate by filamentous fungi].

Selected filamentous fungi--98 strains of 31 genera--were screened for the ability to catalyze 11beta-hydroxylation of 6alpha-fluoro-16alpha-methyl-deoxycorticosterone 21-acetate (FM-DCA). It was established that representatives of the genera Gongronella, Scopulariopsis, Epicoccum, and Curvularia have the ability to activate 11beta-hydroxylase steroids. The strains of Curvularia lunata VKM F-644 and Gongronella butleri VKM F-1033 expressed maximal activity and formed 6lpha-fluoro-16alpha-methyl-corticosterone as a major bioconversion product from FM-DCA. The structures of the major products and intermediates of the bioconversion were confirmed by TLC, H PLC, MS and 1H NMR analyses. Different pathways of 6alpha-fluoro-16alpha-methyl-corticosterone formation by C. lunata and G. butleri strains were proposed based on intermediate identification. The constitutive character and membrane-binding localization were evidence of a 11beta-hydroxylating system in G. butleri, while an inducible character and microsomal localization was confirmed for 11beta-hydroxylase of C. lunata. Under optimized conditions, the molar yield of 6alpha-fluoro-16alpha-methyl-corticosterone reached 65% at a FM-DCA substrate loading of 6 g/L.

[The morphometric analysis of serotonin granules of platelets in migraine].

The serotonin hypothesis of migraine attack is based on a phenomenon of fast and significant decreasing of serotonin granules in blood platelets. The morphometric analysis was used to study area, diameter, length and wideness of platelet serotonin granules in 20 migraine patients during a headache attack and in an interparoxysmal period. We found that the area of serotonin granule surface in platelets was 0.231 +/- 0.595 mcm during the migraine attack that was significantly higher than that during the interparoxysmal period (0.205 +/- 0.037 mcm) and in the platelets of healthy controls (0.190 +/- 0.028 mcm) (n = 10). The study has shown that during migraine attack serotonin granules are undergone also structural changes.

[Bioconversion of C19- and C21-steroids with parent and mutant strains of Curvularia lunata].

Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKMF-644. The products of the transformations were isolated by column chromatography and identified using HPLC, mass-spectrometry (MS) and proton nuclear magnetic resonance (1H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9alpha-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14alpha, while 6alpha-, 6beta- and 7alpha-hydroxylated products were revealed in minor amounts. At the transformation of C21-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11beta-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11beta-hydroxylase activity towards 21-acetate and 17alpha,21-diacetate of cortexolone was obtained. Yield of 11beta-hydroxylated products reached about 90% at the transformation of 17alpha, 21-diacetate of cortexolone using mutant strain M4.

[Involvement of serotonergic system in the pathogenesis of non-motor symptoms of Parkinson's disease].

Platelet serotonin content was investigated in 51 patients with Parkinson's disease and compared to the form and severity of non-motor symptoms - depression, anxiety and cognitive dysfunction. An average level of platelet serotonin was 166,8+/-111,1 ng/ml in parkinsonian patients that was significantly lower (p=0,027) compared to the control group (562,7+/-111,1 ng/ml). The platelet serotonin level in the peripheral blood was inversely correlated with clinical form of disease, cognitive deficits and the level of anxiety and depression.

[Serum-blood serotonin level as a marker of panic attacks severity and effectiveness of their treatment].

Twenty-eight males with psychogenic (emotiogenic) autonomic dystonia with paroxysmal course (panic attacks) have been studied. Serum serotonin (S) concentration has been measured using ELISA. The deficit (147,36+/-69,80 ng/ml) of humoral S has been found compared to a control group (295,18+/-152,38 ng/ml). After the 8-weeks treatment with a SSRIs antidepressant rexetin in dosage 20 mg/day, a distinct clinical effect has been observed. There was a decrease of the number and severity of paroxysms as well as severity of symptoms of psychoautonomic syndrome (the reduction of depression, anxiety and optimization of autonomic regulation). Despite the therapeutic effectiveness of rexetin, serum S concentration has decreased still more to 59,92+/-23,06 ng/ml. The paradoxical phenomenon of growing deficit in the humoral pattern of serotonergic system during the short-term course of SSRIs antidepressant treatment is reviewed as a result of the transfer of neuromediator from the blood to the cerebral structures with the following activation of serotonergic system that provides the therapeutic effect.

[Correction of motor and affective symptoms of parkinsonism by selective serotonine reuptake inhibition antidepressant].

A study included 68 patients with parkinsonism: 43 of them were treated with proflusak (fluoxetin), which efficacy was assessed in the frames of randomized placebo-controlled study. The levels of motor disability, anxiety, depression and serum serotonin (S) concentration were measured. The prevalence of rigidity and hypokinesia caused the increase of the depression level, along with decreasing of S concentration. Severity of tremor was positively correlated with the level of anxiety and blood S. Significant differences between patients receiving proflusak and placebo were found. No changes in the levels of motor-affective symptoms were observed in the placebo group. In patients treated with proflusak, the subjective improvement of health was accompanied by the significant decrease of depression severity and reduction of movement deficit, in particular rigidity and hypokinesia. The blood concentration of S decreased significantly in comparison to the placebo group. Taking into account the mechanism of action of the drug studied (its belonging to SSRIs), these changes may reflect the alterations of serotonergic metabolism in the brain synapses.

[Efficacy of rehabilitation of patients with chronic primary low back pain at the spa Klyuchi using balneopelotherapy and transcranial electrostimulation].

Low back pain caused by vertebrogenic pathology is encountered in 60-80% of adult population. We studied efficacy of balneopeloid factors of Klyuchi health resort (Perm Region) and transcranial electrostimulation (TCE) in 60 patients with primary chronic low back pain. The patients were divided into two groups. Thirty patients of group 1 received balneopelotherapy (sulfide mud), Thirty patients of group 2 were given the same balneopelotherapy plus TCE. Pain relieved and psychovegetative status improved in both groups but the effect was more pronounced in group 2.

[Leucocytes' ability to accumulate and secrete cholesterol is a nonspecific factor of immune inflammation in patients with atherothrombotic stroke].

The aim of the study was investigation of nonspecific inflammation markers in atherosclerotic injury of major arteries of the head (MAH). A complex study, including duplex ultrasonography, CT, measurement of cholesterol and its fractions, has been conducted in 85 patients with atherothrombotic (72 patients) and lacunar (23 patients) stroke. The ability of leucocytes' culture to accumulate and secrete cholesterol in vitro was studied as a nonspecific inflammatory marker of lesion of atherosclerotic MAH. A high level of this index was characteristic of atherothrombotic type of stroke comparing to lacunar stroke and the control group. Furthermore, high levels of this index correlated with severity of atherosclerotic injury of MAH, in particular with plaques. The dynamics of the index after 24-day therapy by statins (vasilip) is presented. We suppose that the protective influence of statins in patients after ischemic stroke may be caused by the effects on the level of immune competent cells including reducing leucocytes ability to accumulate and secrete protein-lipid complexes.

[Method for determining leukocytic lipid-releasing capacity in the diagnosis of mechanisms responsible for atherogenesis in patients with coronary heart disease and ischemic stroke].

The authors have studied the mechanism responsible for neutrophilic accumulation and release of protein-lipid complexes in patients with atherosclerosis and the clinical value of this reaction. They present the results of experimental studies and clinical observations in the groups of patients with ischemic stroke, coronary heart disease, bronchial asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis. Whether the process of neutrophilic and release of protein-lipid can be in vitro modulated in a 3-day culture has been demonstrated. The high diagnostic value of the method for determining the lipid-releasing capacity of leukocytes is shown. Some proteins involving in the formation of pathogenic protein-lipid complexes are identified. There is evidence for the use of new differential criteria for visceral diseases originating from atherosclerosis.

[Quantitative determination of serum serotonin in the diagnosis of tension headache].

A comprehensive psychometric testing and measurement of the serum levels of serotonin by enzyme immunoassay were made before and after treatment with the antidepressant Prodep in 76 patients with chronic tension headache. The patients generally had cephalgia and moderate depression, which were associated with the lower blood concentration of serotonin. A course of antidepressant therapy with Prodep ameliorated headache and depression in the presence of higher serum serotonin levels. Serum serotonin may be regarded as a marker of the severity of pain and depression in patients with chronic tension headache and be an objective indicator of the efficiency of antidepressant therapy.

[Endothelium dysfunction in patients with ischemic stroke].

A vasomotor endothelial function has been studied in 57 patients with ischemic stroke using ultrasound by the reaction of the brachial artery to reactive hyperemia. The duplex scanning of brachyocephalic arteries was conducted. Twenty-four patients had impaired endothelial-dependent vasodilatation. Endothelial dysfunction was associated with cholesterol blood level and smoking. Aging, positive family history and arterial hypertension did not affect this parameter. A level of vasomotor dysfunction correlated with intima-media correlation, the presence of plaques in the carotid arteries and internal carotid artery occlusion. Impaired endothelial-dependent vascular relaxation occurred more frequently in atherothrombotic than in lacunar stroke and in patients with a severe brain damage.

[Serum serotonin in patients with tension headaches].

Tension headache (TH) is one of the most frequent types of idiopathic headaches. The leading role in its pathogenesis is played by depression and dysmetabolism of the neurotransmitter serotonin. The subjects were 100 patients with TH. The examination included study of headache intensity, complex psychometric testing, and immune-enzyme measurement of serotonin serum level, performed before and after treatment with the anti-depressant prodep. All the patients had moderate pain syndrome, depression, and moderate or severe anxiety, which demonstrated negative correlation with serotonin serum level. In particular, the intensity of episodic THs (n = 24) was 52 mm visual analogue scale, reactive anxiety level was as high as 51.08 +/- 4.2, the level of depression was moderate (12.9 Beck scale); serotonin blood level showed a tendency to fall (205.72 +/- 6.74 ng/ml). In 76 patients, suffering from chronic THs with a cephalgia intensity of 62 mm visual analogue scale, high indexes of reactive and personal anxiety (46.81 -/+ 2.68 and 54.2 +/- 3.64, respectively), and high depression level were associated with a prominent decrease of serotonin blood concentration (119.38 +/- 9.42 ng/ml). A course of treatment with prodep led to significant decrease of headache intensity and improved depression, while an increase of serum serotonin level was observed. Thus, serotonin serum level may be used as a marker of pain intensity and the level of depression, and an objective indicator of anti-depressive therapy.

[Clinical course of autonomic nervous system disorders in veterans with consequences of combat mild brain injury]. / Dinamika psikhovegetativnykh rasstroistv u lits s posledstviiami legkoi boevoi cherepno-mozgovoi travmy.

Twenty-two veterans with combat mild brain injury (BI) were studied 7 and 14.9 years after BI. Along with evaluation of clinical symptoms, the authors investigated the autonomic tonus, autonomic background, regulatory brain systems function (cardiointervalography), emotional and personality state measured by Scmiscek-Litman (MMPI-modification) and Spilberger tests. Initial examination identified clinical syndromes, such as autonomic dystonia (70% of the patients), psychopathological syndromes represented by asthenic, affective, neurotic and neurotic-like states (90%), cochleo-vestibular (30%), insomniac (20%) states. Follow-up revealed these syndromes in 100, 100, 45, 20% of the cases, respectively. There was an increase in the dysfunction of nonspecific brain systems, which emerged clinically as a psychoautonomic syndrome in the form of autonomic dystonia with an elevation of sympathetic activity and emotional shift to depressive and hypochondriac disorders.

[Physical factors in rehabilitation of patients with bronchial asthma]. / Fizicheskie faktory v reabilitatsii bol'nykh bronkhial'noi astmoi.

154 patients with moderate severity bronchial asthma (BA) received treatment including two components: electroaerosols with mineral sodium chloride water (Ust-Kachka resort) (group 1) or ward speleotherapy (group 2), standard antiinflammatory medication with beklametasone dipropionate. (BD). A control group was given BD only. Personality traits and vegetative pattern were studied before and after the treatment. General clinical response was the same in all three groups. Normalization of emotional and vegetative abnormalities was faster in groups where physical factors were employed. These findings show perspectiveness of a new line in the treatment of BA--neurorehabilitation.

[Diagnostic significance of the type of in vitro interaction between blood lymphocytes and serotonin in multiple sclerosis]. / Diagnosticheskoe znachenie kharaktera vzaimodeistviia limfotsitov krovi s serotoninom v sisteme in vitro pri rasseiannom skleroze.

The method for laboratory diagnosis of multiple sclerosis (MS)--the loading serotonin test in vitro based on a specific type of interaction between MS patient's blood lymphocytes and serotonin--is suggested. An examination of 56 MS patients, 12 patients with vertebrale nervous system pathology, 33 patients with MS phenocopies and 19 healthy subjects revealed an initial low serotonin rosette-forming cell (RFC) content in MS patients, comparing to all the subjects studied. In contrast to healthy subjects and patients with other neurological disorders, serotonin receptor lymphocyte expression in MS patients does not change during serotonin stimulation in vitro and serotonin RFC number does not increase. The type of interaction is specific and selective, being confirmed in reaction of rosette-formation by serotonin excess and by the results of rosette-formation reaction with other biologically active components (histamine, nor adrenaline). A serotonin reception inhibition, specific for MS, can be determined already in the disease debut, regardless of patient's gender and age, clinical type, disease stage and duration, which proves to be of most importance. The loading serotonin test indexes are significant for differential diagnosis. The method patented in 1999 is realized in vitro, excludes a double blood collection that reduces allergy and infection danger for patients is easy to make and cost-effective.

[Diagnostic value of rosette-forming acetylcholine specific lymphocytes in the blood of myasthenia gravis patients]. / Diagnosticheskoe znachenie soderzhaniia v krovi bol'nykh miasteniei rozetkoobrazuiushchikh limfotsitov spetsifichnykh atsetilkholinu.

In a blood of 37 patients with different forms of myasthenia (M) there was found an increase of a number of rosette-forming lymphocytes specific to acetylcholine in a specific rosette-forming reaction with acetylcholine erythrocytes' diagnostic kit. A value of this index correlated with a duration of the disease, with a degree of its generalization, with a degree of its severity and with the type of myasthenic process' course. Meanwhile, it wasn't depended on the age of the patients. In a blood of both healthy individuals and the patients with some other neurologic diseases a number of rosette-forming lymphocytes specific to acetylcholine was considerably lower. A number of the lymphocytes specific to acetylcholine was increased even in the debut of M and remained altered during all the period of the disease. That may serve as an additional diagnostic marker of M in any age, at different character and severity of the disease, in any phase of this pathology.