A novel method of HPV genotyping using Hybrid Capture sample preparation method combined with GP5+/6+ PCR and multiplex detection on Luminex XMAP.

A novel DNA detection assay comprising Hybrid Capture sample preparation, GP5+/6+ PCR with modifications and Luminex 100 detection was developed and applied to genotyping of human papillomavirus (HPV) in cervical samples. Target-specific sample preparation was performed using magnetic beads conjugated with Hybrid Capture (HC) antibody. DNA-RNA hybrids were formed between DNA target and RNA probes and captured on HC-beads. DNA on magnetic beads was amplified without elution using consensus GP5+/6+ PCR and then genotyped on Luminex beads using hybridization probes for the 17 high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 and an internal control. This new sequence-specific Hybrid Capture sample preparation is fast, efficient and allows direct HPV genotyping by PCR. Compared to traditional non-sequence-specific sample preparation methods, HC sample preparation demonstrated slightly better detection of multiple HPV infections. The clinical utility of this method was demonstrated on cervical samples positive for HR HPV by the Hybrid Capture 2 (hc2) screening assay.

Synovial Sarcoma Microvesicles Harbor the SYT-SSX Fusion Gene Transcript: Comparison of Different Methods of Detection and Implications in Biomarker Research.

Background. Synovial sarcoma is an aggressive soft-tissue malignancy. This study examines the presence of the SYT-SSX fusion transcript in synovial sarcoma microvesicles as well as its potential role as a biomarker for synovial sarcoma. Patients and Methods. Microvesicle release of synovial sarcoma cells was examined by transmission electron microscopy. RNA-content was analyzed by qPCR, nested PCR, nested qPCR, and droplet digital PCR to compare their sensitivity for detection of the SYT-SSX fusion gene transcript. Whole blood RNA, RNA of mononuclear cells, and microvesicle RNA of synovial sarcoma patients were analyzed for the presence of the fusion gene transcripts. Results. Electron microscopic analysis revealed synovial sarcoma cells releasing membrane-enclosed microvesicles. In vitro, the SYT-SSX fusion gene transcript was detected in both synovial sarcoma cells and microvesicles. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA. In contrast, the fusion gene transcript was not detected in peripheral blood cells and microvesicles of synovial sarcoma patients. Conclusion. Synovial sarcoma cells release microvesicles harboring the SYT-SSX fusion transcript. Nested qPCR proved to be the most sensitive in detecting the SYT-SSX fusion gene mRNA; however, more sensitive assays are needed to detect cancer-specific microvesicles in the peripheral blood of cancer patients.

Determination of tRNA(Phe) recognition nucleotides for phenylalanyl-tRNA synthetase from Thermus thermophilus.

The tRNA(Phe) recognition nucleotides for phenylalanyl-tRNA synthetase from an extreme thermophile Thermus thermophilus were investigated. Using yeast tRNA(Phe) T7 transcripts with various point mutations it was shown that four recognition points (G34, A35, A36 from anticodon and A73 from acceptor stem) are important for aminoacylation at 37 degrees C. In the case of the 73rd discriminator base A----U, but not A----C, substitution suppresses aminoacylation. Position 20, which proved to be essential for all phenylalanyl-tRNA synthetases so far studied, is not involved in the recognition of tRNA(Phe) by the T thermophilus enzyme.

Detection of telomerase activity utilizing energy transfer primers: comparison with gel- and ELISA-based detection.

We have developed a closed-tube format telomeric repeat amplification protocol (TRAP) assay for direct quantification of telomerase activity within the PCR vessel. The assay utilizes energy transfer (ET) primers, which emit fluorescence only upon incorporation into PCR products. This novel ET primer system (Amplifluor primers) has major advantages over existing detection methods because it eliminates the need for post-PCR processing and thus reduces greatly the risk of carryover contamination and the time required for the sample analysis. The assay is as sensitive, specific and quantitative as the polyacrylamide gel-based or ELISA-based TRAP assay.

In situ amplification using universal energy transfer-labeled primers.

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used.

[Effect of nucleotide replacement on the effectiveness and specificity of the SP6 promotor]. / Vliianie nukleotidnykh zamen na éffektivnost' i spetsifichnost' SP6-promotora.

The interaction of SP6 RNA-polymerase with its promoter (sequence; see text) where +1 is the initiation site, was investigated. The substitution of C for A at +2 position and T for A at +3 creates the AluI-site (-2-1+1+2). The efficiency of the promoter changed in this way is comparable with that of the natural one. The introduced restriction site permits to insert the gene precisely at the initiation site and the synthesis of the transcripts with the authentic 5'-ends. The influence of the nucleotide substitution within -5-10 region of SP6-promoter on its interaction with SP6 and T7 RNA-polymerases was investigated. A new promoter, able to initiate the transcription with both SP6 and T7 RNA polymerases, was obtained.

[Unprimed synthesis of poly (d(A-T)), catalyzed by a preparation of Escherichia coli DNA polymerase I]. / Issledovanie prirody bezmatrichnogo sinteza d(A-T)-sopolimera, katalizirchemogo preparatami DNK-polimerazy I Escherichia coli.

The initial events of the de novo synthesis of poly[d(A-T)], catalyzed by preparations of E. coli DNA-polymerase I, were investigated. The data provide evidence that deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase), the enzyme which is able to catalyze unprimed polymerization of dNDP, participates in the process of initiation. This conclusion is based on the following data: 1) preincubation of E. coli DNA-polymerase I preparation with dADP and dTDT abolishes a lag-period in the poly[d(A-T)] synthesis; 2) dithiothreitol and N-ethylmaleinide, inhibitors of dNDP-transferase, inhibit de novo synthesis of [d(A-T)]-copolymer by preparations of E. coli DNA-polymerase I but do not effect primed synthesis ensured by this enzyme. High concentration of the substrate have similar effect. Using two-dimentional thin-layer chromatography and microcolumn chromatography on TEAE-cellulose we have shown that preliminary incubation of DNA-polymerase I preparations with dADP and dTDP results in the synthesis of short oligonucleotides (from di- to decanucleotides). Hydrolysis of these oligonucleotides with dilute sulfuric acid demonstrates that among the reaction products prevail oligoadenylates and oligothymidylates, but an appreciable amounts of heterooligomers including oligo[d(A-T)] were revealed as well. The model of so called de novo synthesis of regular polynucleotides is proposed, according to which dNDP-transferase, an accompanying enzyme in the preparations of DNA-polymerase I E. coli, is carrying out the synthesis of short oligonucleotides which form template-primer complexes repeatedly replicated by the DNA-polymerase I E. coli.

[Separation of the enzyme catalyzing polymerization of deoxyribonucleoside diphosphates from preparations of E. coli DNA-polymerase I]. / Vydelenie fermenta kataliziruiushchego polimerizachiiu dezoksinykleoziddifatov, iz preparatov DNK-polimerazy I E. coli.

The enzyme which catalyses template independent synthesis of polydeoxynucleotides from deoxynucleoside diphosphates was separated from E. coli DNA polymerase I by DEAE-cellulose chromatography followed by ultrafiltration through the M-50 Amicon filter. The ultrafiltration data indicate that the molecular weight of the enzyme is not higher than 50,000. The enzyme is not able to use deoxynucleoside triphosphates, ribonucleoside di- or triphosphates as substrates for the polymerization. The reaction of template independent polymerization proceeds with a lag period varying from 2 to 20 hours (for different preparations of enzyme) and is activated by Mg2+ (the optimal concentration 1-2 . 10(-3) M). The pH optimum of the reaction is at 8.5. The optimal concentration of deoxyribonucleoside diphosphates is 10(-3) M, and its increase strongly inhibits polymerization. The enzyme was supposed to be called deoxynucleoside diphosphate: olygonucleotide deoxynucleotidyltransferase (catalyzing polymerization without template). The presence of the enzyme in the preparations of E. coli DNA-polymerase I can explain the ability of the latter to catalyze the untemplated synthesis of poly dG : poly dC.

[Cleavage of synthetic RNA-DNA hybrids with restriction endonucleases BamH1 and Sau3A]. / Rasshcheplenie sinteticheskikh RNK-DNK-gibridov éndonukleazami restriktsii BamH1 i Sau3A.

Synthetic oligodeoxyribonucleotides, containing one or two ribonucleotides in the recognition sequence, and RNA--DNA hybrids were tested for their activity in cleavage with BamH1 and Sau3A endonucleases. The replacement of dG with G in the first position of BamH1-site (GGATCC) of one of the chains does not affect the rate of the BamH1 hydrolysis. The similar heteroduplex, containing G residue in the second position, displays a decreased rate of the BamH1 hydrolysis of the modified strand and, to a lesser extent, of the unmodified complementary strand. Oligodeoxyribonucleotides in complex with oligoribonucleotides can be cleaved with the excess of BamH1 and Sau3A, oligoribonucleotides remaining intact.

[The effect of beta-casomorphin-7 on the level of food and defense motivations in different types of learning]. / Vliianie beta-kazomorfina-7 na uroven' pishchevoi i oboronitel'noi motivatsii pri razlichnykh vidakh obucheniia.

The effects of intraperitoneal administration of the heptapeptide beta-casomorphin-7 (beta-C-7) on learning were investigated in white rats using T-maze, active and passive avoidance tests. The substance injected in the dose of 5 mg/kg 5 min prior to training accelerated the acquisition of food-reinforced task increasing the number of correct trials in T-maze. The same injection in the dose of 1 mg/kg impaired the acquisition of avoidance tasks. The heptapeptide didn't influence food motivation and consolidation of memory trace. The data obtained support the idea that beta-C-7 attenuated manifestations of defensive motivation.

[Comparative characteristics of the lipoprotein spectrum in hypercholesterolemia in 40- to 59-year-old men living in Moscow and Kharkov]. / Sravnitel'naia kharakteristika spektra lipoproteidov pri giperkholesterinemii u muzhchin 40--59 let, prozhivaiushchikh v Moskve i Khar'kove.

Total cholesterol, triglycerides and high- and low-density lipoprotein cholesterol were measured in a study of 40-59-years-old male populations in Moscow and Kharkov. Hypercholesterolemic individuals were identified, using standard criteria, in both cities. Although mean lipid parameters were similar in individuals with second-type hyperlipoproteinemia from Moscow and Kharkov, the pattern of hypercholesterolemia was different in terms of the lipoprotein spectrum. It is concluded that detailed differentiation of second-type hyperlipoproteinemia is essential for the identification of major mechanisms of the atherogenic potential of hypercholesterolemia in different populations.

[Features of blood lipid spectrum in a schoolchildren's population in Kharkov]. / Osobennosti lipidnogo spektra krovi v populiatsii shkol'nikov Khar'kova.

The blood lipid and lipoprotein spectrum was biochemically studied in 580 pupils aged 12-16 years. The normal value ranges were found for total cholesterol (TC), triglycerides (T), high density lipoprotein (HDL) and low density lipoprotein in the Kharkov schoolchildren population. The upper limits of lipid lipoprotein levels enabled patients with hyperlipoproteinemias to be identified in the studied age groups. The Kharkov children were demonstrated to have high TC and T levels and relatively low HDL cholesterol, i. e. they had unfavourable characteristics as to atherogenesis. The findings suggest that the factors that influence the formation of atherogenic dyslipoproteinemias should be further studied and the preventive measures aimed at normalizing the lipid spectrum should be implemented.

[The use of reaferon in treating patients with protracted viral hepatitis]. / Primenenie reaferona v lechenii bol'nykh zatiazhnym virusnym gepatitom.

The paper reports the results of a randomized clinicoimmunological trial of domestic recombinant alpha 2-interferon (reaferon) in 30 patients with protracted viral hepatitis (27 subjects had viral hepatitis B and 3 patients HBV + HDV-coinfection). The reaferon treatment started on the disease week 7-8 and lasted for 2 months (1 x 10(6) IU, once a week, i.m.). VHB patients benefited from reaferon treatment as evident from improved clinical and laboratory indices, cases of complete cure and the agent elimination. In a protracted course of HBV + HDV coinfection reaferon in the above doses failed to produce any effect.

[The apoB genotype and the efficacy of hypolipidemic therapy]. / Genotip apoB i éffektivnost' gipolipidemicheskoi terapii.

Several men were examined for association between restriction fragment length polymorphism (RELP) Xba I (exon 26) number of tandem repeats in 3'-hypervariable region of the apolipoprotein-B gene and serum levels of cholesterol and triglyceride. These two types of polymorphism were studied. An association of the Xba I site and alleles containing more repeats in the 3'-hypervariable region with higher cholesterol and triglyceride was observed. 32 patients with CHD aged 24-56 years were examined. All the patients are males with clinical picture of CHD (stable angina pectoris of II-III functional classes) and dyslipoproteinemia of II a, II b and IV types by D. S. Fredrickson. Xba-I polymorphism of apo-B gene was detected by DNA polymerase reaction method. The following Xba-I genotypes were distinguished: X1X1 (absence of Xba I site); X1X2 (heterozygosity on Xba I site) and X2X2 (homozygosity on Xba-I site). Lipantil (fenofibratte) was prescribed in a dose of 300 mg daily after meals (100 mg three times a day). Data obtained show that DNA polymorphism of apo-B gene not only influences plasma lipids concentration but also determines effectiveness of hypolipidemic therapy. Hypolipidemic effect of lipantil depends on Xba-I site presents in apo-B gene and is significantly expressed in homozygous patients with X1X1 genotype.

[Therapy of postresuscitation behavioral disorders with mexidol and kyotorphin]. / Korrektsiia postreanimatsionnykh narushenii povedencheskikh reaktsii meksidolom i kiotorfinom.

A complex of behavioral tests revealed diminished anxiety, increased locomotor and exploratory activities, and changes in different learning tests in resuscitated animals. Mexidol alone and in combination with kyotorphin exhibited antistressogenic and nootropic activities, and led to a compensation of ischemic lesions in rats. It can be stated that a the injection of mexidol in combination with kyotorphin yielded better results due to its neuroprotective effect in the CA1 and CA4 fields of the hippocampus.

[Surgical treatment of atherosclerotic occlusion of the femoral, popliteal and tibial arteries]. / Khirurgicheskoe lechenie ateroskleroticheskikh okkliuzii bedrennoi, podkolennoi i bertsovykh arterii.

An analysis of results of the treatment of 182 patients with atherosclerotic occlusions of femoral, popliteal and tibial bones has shown the necessity of an individual approach to choosing the method of restorative operation on the vessels. More favourable remote results were obtained after autovenous shunting "in situ" and by the method of vein reversion.

[Functional-morphologic evaluation of the effect of the regulatory peptide kyotorphin on the status of the CNS in the post-resuscitation period]. / Funktsional'no-morfologicheskaia otsenka vliianiia reguliatornogo peptida kiotorfina na sostoianie TsNS v postreanimatsionnom periode.

Functional and morphological analysis of changes in white rat CNS was made throughout 4 months after 12 minute interruption of circulation. Within 3 months after clinical death and resuscitation the rats' neurological status was normal except some abnormalities in emotions and motives especially noticeable under stress. Moreover, neurons in the hippocampus and cerebellum were destroyed. A single intranasal 0.05 mg/kg dose of kyotorphin--a peptide isolated from the brain of winter-sleeping animals--given 30 min after beginning of resuscitation increased survival, accelerated restoration of neurological status, normalized emotional reactivity, orientation and search behavior, prevented death of neurons in the hippocamp and cerebellum.

[Effect sandostatin on functional and structural recovery of the central nervous system after clinical death]. / Vliianie sandostatina na funktsional'no-strukturnoe vosstanovlenie tsentral'noi nervnoi sistemy posle klinicheskoi smerti.

A behavior examination set was used to reveal a decreased anxiety as well as increased locomotor and exploratory activities and changes in resuscitated animals undergoing different learning tests. A single injection of sandostatin--an analogue of regulatory peptide somatostatin--was found to improve the structural and functional recovery of the central nervous system after a 12-minute cardiac arrest.