Babesia DNA detection in canine blood and Dermacentor reticulatus ticks in southwestern Siberia, Russia.

Babesia infection was studied in 21 blood samples of dogs with symptoms of babesiosis and among 72 Dermacentor reticulatus and 70 Ixodes persulcatus ticks from southwestern Siberia, Russia. Babesia DNA was detected by hemi-nested PCR based on the 18S rRNA gene with subsequent direct sequencing. All of the analyzed canine blood samples and three D. reticulatus, but none from I. persulcatus ticks studied were shown to contain Babesia DNA. Nucleotide sequences of the Babesia 18S rRNA gene fragment of 354 bp long for all 24 positive samples appeared to belong to the subspecies Babesia canis canis and differed only at three positions. The Babesia nucleotide sequences from 17 canine blood samples and from one D. reticulatus tick were identical to each other and to previously known B. canis canis from canine blood in Slovenia. Four canine blood samples and the second tick sample contained a mixture of two nucleotide sequences previously found in canine blood. B. canis canis nucleotide sequence from the third tick differed in the unique nucleotide transition and could correspond to a new genetic variant. Thus, the main etiological agent of canine babesiosis in Novosibirsk region is B. canis canis, and D. reticulatus, but not I. persulcatus, ticks could serve as a vector of this infectious agent. To our knowledge, this is the first report of the B. canis canis nucleotide sequences from ticks.

Phosphorylation of tick-borne encephalitis virus NS5 protein.

The largest tick-borne encephalitis virus (TBEV) non-structural protein NS5 (100 kDa) is believed to be involved in RNA replication. The protein is phosphorylated in infected cell extracts in the presence of [gamma-32P]ATP, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using monoclonal antibodies raised against TBEV NS5 protein. Radioactive labeling of NS5 in cellular extracts at an early stage post-infection is higher than at 24 h post-infection. Incubation of immunoprecipitates of NS5 protein with [gamma-32P]ATP in the presence of Mg2+ resulted in the phosphorylation of TBEV NS5 protein and of immunoglobulins. Phosphoamino acid analysis demonstrated that NS5 contains phosphoserine, but not phosphothreonine, or phosphotyrosine.

Tick-borne encephalitis virus NS3 gene expression does not protect mice from homologous viral challenge.

Tick-borne encephalitis virus (TBEV) NS3 gene has been subcloned into the expression vector pcDNAI and expressed in eukaryotic cells. Immunization of mice with the recombinant plasmid pcDNAI-NS3 induced antibodies against NS3 protein but did not protect from viral challenge.

[Expression of the NS1 gene of tick-borne encephalitis virus in gram-negative bacteria from the mouse nasopharynx]. / Ekspressiia gena NS1 virusa kleshchevogo éntsefalita v gramotritsatel'nykh bakteriiakh--obitateliakh nosoglotki myshei.

Bacteria were isolated from the nasopharynx of BALB/c mice and electroporated with pUR290(NS1)2 containing two copies of tick-borne encephalitis virus (TBEV) strain Sofjin NS1 under the control of the lac promoter. The plasmid persisted in transformants for at least ten passages. The NS1 gene expression was detected in Gram-negative enterobacteria via immunoblotting with monoclonal antibodies against TBEV nonstructural glycoprotein NS1. Recombinant NS1 was detected in bacterial cells and in the culture medium. Intranasal immunization with recombinant bacteria activated production of antibodies against NS1 in serum of BALB/c mice. The humoral immune response to NS1 failed to protect immunized mice from a TBEV challenge.

[Transfection of bacterial cells by DNA of phage M13 entrapped in phospholipid vesicles]. / Transfektsiia bakterial'nykh kletok zakliuchennoi v fosfolipidnye vezikuly DNK faga M13.

The possibility of transfection of bacterial cells by phage M13 DNA entrapped into phospholipid vesicles (liposomes) has been studied. Two types of liposomes differing in size were used. Entrapped DNA was transferred by liposomes into Ca2+-treated E. coli cells. Efficiency of the transfection in the case of small (ca. 400 A) liposomes was 2--3 orders of magnitude higher than that of free DNA extracted from such liposomes.

[Highly selective affinity labeling of DNA-dependent RNA-polymerase of bacteriophage T7]. / Vysokoselektivnoe affinnoe mechenie DNK-zavisimoi RNK-polimerazy bakteriofaga T7.

T7 phage RNA polymerase was affinity labelled in the presence of its promoter by treatment with an ATP gamma-derivative (a phosphoamide obtained from 4-(N-chloroethyl, N-methyl)aminobenzylamine, or one of esters obtained from 2-methoxy-4-formylphenol, 4-formylphenol, and 2[N-(4-formylphenyl), N-methyl]-aminoethanol) followed by addition of [alpha-32P]GTP. The most efficient labelling took place with the alkylating phosphoamide reagent.

[Highly selective affinity labeling of DNA-dependent RNA-polymerase II from human placenta]. / Vysokoselektivnoe affinnoe mechenie DNK-zavisimoi RNK-polimerazy II iz platsenty cheloveka.

RNA polymerase II from human placenta was affinity labelled in crude preparation using two-step technique, which includes treatment of the enzyme with an aldehyde-containing reactive analogue of ATP, ADP or AMP in the presence of poly[d(A-T)] followed (after borohydride reduction) by the elongation of the attached label with [alpha-32P]UTP. A polypeptide of the molecular mass ca. 140 kDa proved to be the labelling target. No labelling was observed in the absence of poly[d(A-T)] or the reagent or in the presence of alpha-amanitin. All the results suggest the attachment of the affinity reagents to the second-largest subunit of the human RNA polymerase II, which therefore takes part in the initiation substrate's binding.

[Determination of ureaplasma DNA by the competitive and multiplex PCR]. / Opredelenie kolichestv DNK ureaplazm s pomoshch'iu konkurentnoi i multikompleksnoi PTsR.

Quantities of Ureaplasma spp DNA were determined by the methods of competitive and multiplex polymerase chain reactions (PCR). The results obtained by each of the methods were shown to be compatible. It was established that the number of Ureaplasma spp. cells per one epithelial cell in the female cervical canal varied from 0.05 to 10.

[Isolation of a complex of tick-borne encephalitis viral proteins by affinity chromatography using monoclonal antibodies to proteins NS5 and NS3]. / Vydelenie kompleksa belkov virusa kleshchevogo éntsefalita metodom affinnoi khromatografii s ispol'zovaniem monoklonal'nykh antitel k belkam NS5 i NS3.

Affinity chromatography of lysates of continuous porcine embryo cells infected with tick-borne encephalitis (TBE) virus on sepharose with immobilized monoclonal antibodies to TBE virus proteins NS5 and NS3 results in isolation of a stable protein complex. This complex contains viral proteins NS5, NS3, p49, and, probably, two more cell proteins. This complex is not detected at the early stage of infection, and 24 h after infection its structure does not depend on the specificity of immobilized monoclonal antibodies used in affinity chromatography and on the time elapsed after the infection. Immunoprecipitates of infected cells phosphorylated TBE virus protein NS5 in vitro, but possessed no RNA-polymerase activity. Elution of the complex with buffers with pH 3.5 or 11.1 or with 2M urea failed to purify the active replicase. The complex of viral and cellular proteins isolated by affinity chromatography on different immunoadsorbents did not possess RNA-polymerase activity because of inactivation or absence of additional subunit(s).

[Interaction of tick-borne encephalitis virus RNA with proteins]. / Vzaimodeîstvie RNK virusa kleshchevogo éntsefalita s belkami.

Binding of tick-borne encephalitis (TBE) virus RNA to proteins was studied by 3 methods: gel retardation, RNA-protein cross-linking under UV light, and RNA-protein blotting. Two cellular proteins with molecular weights about 30 and 22 kDa were detected in the nuclear fraction of two cell strains (PS and RH) and in blood mononuclear cells of patients with TBE. Weak interaction between the studied RNA and viral proteins can result from trace amounts of TBE virus proteins in infected cells and by lack of additional binding factors. Bacterial super-products and affinity chromatography for isolation of native glycoproteins were used to increase the amount of viral proteins. Purified E and NS1 proteins did not react with viral RNA, while incubation with recombinant nonstructural TBE virus NS3 protein led to a shift in the mobility of TBE virus RNA in gel in the absence of cross-linking under conditions of UV exposure after addition of heparin. Poor binding of nonstructural TBE virus NS3 protein is based on electrostatic binding. Oligoribonucleotide 14 nucleotide residues long corresponding to 5'-terminal of TBE virus genome did not react with NS3 protein, probably because of its small size or absence of certain sequences.

[A comparison of the immune response induced by DNA or by an inactivated vaccine against tick-borne encephalitis]. / Sravnenie immunnogo otveta, indutsirovannogo DNK ili inaktivirovannoi vaktsinoi protiv kleshchevogo éntsefalita.

BALB/c mice were immunized with recombinant plasmid DNA pSVK3-ENS1 and pcDNAI-NS3 containing, respectively, genes E-NS1 and NS3 of tick-borne encephalitis (TBE) virus. Antibodies to TBE virus proteins were detected in the blood sera of the immunized animals by the method of the enzyme immunoassay. Though the titers of virus-specific antibodies in the sera of mice immunized with protein vaccines exceeded those registered after immunization with DNA vaccines, essential protective immunity was observed after the use of both vaccines.

[The use of internal controls of different lengths in the detection of Chlamydia trachomatis DNA by the polymerase chain reaction method]. / Ispol'zovanie vnutrennikh kontrolei razlichnoi dliny pri detektsii DNK Chlamydia trachomatis metodom polimeraznoi tsepnoi reaktsii.

To detect C. trachomatis DNA, the polymerase chain reaction (PCR) with the use of primers corresponding to variable sites of rRNA gene 16S was carried out. As the positive control of the reaction, the amplification fragment of gene 16S of rRNA, cloned in the plasmid vector and having the length of 530 nucleotide pairs (n.p.), was used. On its basis 2 kinds of the internal control of the reaction were obtained with the deletion of 110 n.p. (pMOS-Chl420) and the insertion of 930 n.p. (pMOS-Chl1460) within the cloned amplification fragment. The study revealed that the addition of the DNA of pMOS-Chl420 or pMOS-Chl1460 into the reaction mixture did not affect the sensitivity of PCR (0.02 pg of bacterial DNA in the sample) in the detection of C. trachomatis DNA isolated both from the culture of bacterial cells and from clinical samples. But in some cases of the amplification of the DNA of internal control pMOS-Chl420, but not pMOS-Chl1460, was observed in the presence of DNA obtained from clinical samples. It was supposedly linked with a higher sensitivity of Taq DNA-polymerase to the action of inhibitors in the synthesis of high-molecular DNA fragments. The observed high frequency of the inhibition (17%) of PCR makes it expedient to carry out this reaction with the use of the internal control.

[Level of colonization by Ureaplasma urealyticum of definite biovars in a group of women with different clinical symptoms]. / Uroven' kolonizatsii ureaplazmami opredelennykh biovarov v gruppakh zhenshchin s razlichnymi klinicheskimi simptomami.

To evaluate the level of U. urealyticum colonization of female urogenital tract, the method of the multiplex polymerase chain reaction (PCR) in the presence of two pairs of primers, corresponding to genes controlling U. urealyticum 16S rRNA and unique human osteopontin was used. The study of 93 clinical specimens showed no correlation between high colonization level and the presence of definite clinical manifestations of U. urealyticum infection. The determination of ureaplasmic biovars was carried out by the method of PCR in the presence of 3 primers corresponding to the multiple-banded antigen (MBA) gene. Biovar parvo was detected in 85% of the specimens, biovar T960 in 11% and both biovars were detected in 4% of the specimens. The biovar distribution in the groups of women with different clinical symptoms was approximately similar. U. urealyticum of biovar T960 occurred more frequently (33% of the specimens) only in a group of women with vaginal discharge characteristic of inflammation.

[Laws (patterns) and principles of the Armed Forces medical support]. / O zakonakh (zakonomernostiakh) i printsipakh meditsinskogo obespecheniia vooruzhennykh sil.

The modern ideas of methodological, philosophic and subject aspects of military medicine laws and troops medical support principles are described. Periodization of occurrence and application of the Russian Armed Forces medical support organizational principles is presented. The classification of organizational principles according to 5 signs is given.

[Clinical and statistical patterns of the hearing system lesions caused by explosive mine trauma]. / Kliniko-statisticheskie zakonomernosti porazhenii slukhovoi sistemy pri minno-vzryvnoi travme.

Mine-explosive trauma has become the predominant type of pathology in the structure of surgical sanitary losses. In 70-75% of the cases it is combined with acoustic injury that requires the improvement of diagnosis and prediction of acoustic injuries at the stage of qualified medical care. In 772 casualties with mine-explosive trauma admitted to the different departments of Kabul Central Hospital the conditions of explosive injury were identified and statistical analysis of acoustic injury dependence on the above-mentioned conditions was conducted. The quantitative patterns obtained could be used in prognosis of the possible value and structure of acoubarotraumatic losses in the mine-explosive injuries.

EBV-based plasmid DNA rearrangements after transfection of eukaryotic cells.

The cDNA encoding influenza virus (A/Udorn/307/72 strain) M2 protein was subcloned into the EBV-based vector pREP9. Three continuous kidney cellular lines of different origin were transfected with recombinant plasmid pREP9-M2. One and 5 months after transfection plasmid DNA rearrangements were detected by means of restriction analysis of recovered plasmids and their hybridization with an influenza-virus-specific radioactive probe. Deletions were the most frequent type of pREP9-M2 mutations. PCR with primers corresponding to cellular genome and plasmid DNA followed by Southern blot analysis with the [(32)P]-labeled M2-fragment allowed host DNA rearrangements to be revealed in transfected cells.