[Analysis of the Gamete-Fusion Genes in the Haploid-inducing ZMS-P Maize Line].

This article is devoted to the study of the double fertilization mechanism in plants, in particular of the maize gamete membrane fusion genes. We detected and analyzed for the first time gamete-fusion genes in the maize genome. Using the BLAST program, we searched for the hap2 gene (generative cell specific 1 (gcs1)) homologs from Arabidopsis in the maize genome. The ZM_BFb0162K03 maize transcript was found, which had 67% identity to the Athap2 gene and contained a conserved region similar to the Athap2 gene fragment. In mRNA samples from the haploid-inducing and control maize lines, an PCR was conducted by using primers specific to the ZM_BFb0162K03 sequence fragment. Sequences of the PCR products from a fragment (1467 bp) of the Zm_hap2 gene of the haploid-inducing and the control maize lines were identical and also were identical to the maize sequences from the GenBank (ZM_BFb0162K03). PCR products (656 bp region of Zm_hap2) for the ZM_BFb0162K03 (1925 bp) maize sequence were observed for the cDNA of pollen grains, ovary, leaves, and roots of the haploid-inducing and control maize lines. Using the Blastx program, we found significant homology of the maize translated proteins to the GEX2, TET11, and TET12 proteins, involved in Arabidopsis gamete-fusion contacts.

[Analysis of Proliferation and Survival of Agrobacteria after Inoculation of Maize Pistil Filaments].

While the authors have previously developed a method of pistil filament treatment with Agrobacterium cells during blossoming for the transformation of maize generative cells, the mechanism for bacterial T-DNA penetration into the embryo sac remained unknown. This article analyzes the possibility of agrobacterial penetration into the maize embryo via pollen tubes. Microbiological, PCR, and GUS techniques were used to confirm that agrobacteria could spread for up to 20 cm from the sie of inoculation and were detected in maize embryo tissues as aerly as 24 h after inoculation, while they were not revealed after 5-13 days.

[CLINICAL SIGNIFICANCE OF CARDIAC BIOMARKERS INCREASING AND THEIR INTERRELATIONS IN SURGERY WITH CARDIO-PULMONARY BYPASS.]

BACKGROUND: Levelfor cardiac troponin I (TrI) and MB-fraction of creatine kinase (CKMB) increases in cardiomyocyte necrosis, and B-type natriuretic peptide (of BNP) increasing reflects ventricular overload. THE AIM: to study the dynamics of BNP, TRI and CKMV in myocardial revascularisation with cardio-pulmonary bypass and to evaluate the clinical significance of these biomarkers elevated levels and establishing the relationship between BNP and markers of myocardial damage in the perioperative period Materials and methods. The study included 52 patients aged 62.5 (54.75; 70) years. Biomarkers concentrations was determined by immunofluorescence. RESULTS: The initial value of BNP were 57.9 (38.675;88.5) pg/ml, and then increased (p<0,01): at the end of the operation up to 91.75 (59.6;132.75) pg/ml, at 1st day following surgery - up to 260 (157;407) pg/ml, and at 2nd day - up to 184 (115.25;274.5) pg/ml. TrI and CKMV increased (p<0,01) up to 0.95 (0.4175;1.4525) ng/ml and up to 13.1 (5.575;15.525) U/L at the end of surgery, and up to 1,355 (0.76;3.8) ng/ml and 10.5 (5;18.325) U/L at thr Istpostoperative day. Preoperative BNP level and TrI level at the end of surgery were the predictors (p

[Prophylaxis of blood loss and allogenic blood transfusion with fibrinolysis inhibitors produced in Russian Federation in patients undergoing cardiac surgery].

Significant bleeding during cardiac surgery, enough to cause re-exploration and/or blood transfusion, increases morbidity and mortality. Bleeding after cardiopulmonary bypass is related to multiple factors: endothelial dysfunction, thrombocytopenia, dilution coagulopathy. Hyperfibrinolysis is one of the important contributors to increased bleeding. To compare the effect of aprotinin with the effect of lysine analogues (aminomethylbenzoic acid and epsilon aminocaproic acid) examined 63 patients were divided into three equal groups depending on the product used for the prevention of activation of fibrinolysis. Data from rotational thromboelastometry measurements (Rotem Gamma Pentapharm, Germany), blood loss and transfusion were collected. The results confirm that used in the study antifibrinolytics: the lysine analogues aminomethylbenzoic acid and epsilon aminocaproic acid prevent hyperfibrinolysis after cardiopulmonary bypass as well as aprotinin.

[Coagulation profiles during cardiac surgery].

OBJECTIVE: To evaluate patients' hemostasis after cardiac surgery using thromboelastometric and impedance aggregometry. MATERIALS AND METHODS: 66 patients were examined intraoperatively. Comparison group included 45 blood donors. Hemostasis was tested for thromboelastometricRotem Gamma with the assessment of external (exTem) and internal (inTem) pathways of coagulation tests performed detection of heparin (hepTem) and cytochalasin-D-inactivation of platelets (fibTem) to assess the level of fibrinogen. Collagen-induced platelet aggregation was determined in an aggregometer CHRONO-LOG (USA). RESULTS: Significant deviations of the parameters of hemostasis were detected in 52 of the 66 studied patients. In group-1 (23 patients) revealed a residual effect of heparin. The effect manifested prolongation CT (clotting time) inTem to an average of 241 +/- 15 s, compared with CT hepTem--181 +/- 7. Patients in this group were in need of additional administration of protamine sulfate. Postoperative bleeding and resternotomia were observed in 3 patients of group-1. In group-2 (25 patients) CT inTem was 216 +/- 21 with significantly fewer CT hepTem (272 +/- 26). The data indicated excess of protamine sulfate. Platelets aggregation decreased compared to the norm. According to the obtained results, the addition of protamine sulfate is not required, however, in 7 cases the protamine sulfate was administered in a dose of 8.9 +/- 0.8 mg in 6 cases resternotomiya required. In the third group (n = 6) bleeding was observed in 4 patients. The difference in CT-hepCT was significant. Significant variations were revealed in the tests of the activity of the extrinsic pathway of coagulation and cytochalasin-D-induced inactivation of platelets: exMCF- 42 +/- 2 mm (normal 57 +/- 15 mm), fibMCF 5.0 +/- 0.3 mm (norm 12.8 +/- 4.3 mm). The concentration of platelets and their aggregation activity was sharply reduced. Disorders of hemostasis in the third group, designated as dilution coagulopathy. CONCLUSION: Turning thromboelastometric and impedance aggregometry in the study of the coagulation profile of patients undergoing cardiac surgery in postperfusion period brings valuable information and allows a differentiated treatment of hemostasis disorders.

Protein apparatus for horizontal transfer of agrobacterial T-DNA to eukaryotic cells.

This review analyzes agrobacterial virulence proteins and recipient cell proteins involved in horizontal transfer of a T-DNA-protein complex. Specifically, it considers the early stages of the interactions of partners (signal exchange, attachment, close contact); T-DNA release from bacterial cells; channel formation for the transfer of ssDNA between the partners; transfer of agrobacterial T-DNA through the membrane, cytoplasm, and nuclear membrane of the recipient cell and its incorporation into the recipient cell genome. It further discusses possible pathways of agrobacterial ssDNA transfer to the recipient cells. In particular, the possible role of T-pili and VirE2 protein during conjugative transfer of agrobacterial ssDNA between donor and recipient cells is discussed.

Supramolecular complexes of the Agrobacterium tumefaciens virulence protein VirE2.

Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2 proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution in the form of complexes of 2-4 protein molecules of 12-18 nm size. We used computer methods to design models of complexes consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering. We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial length of the ssDNA.

[Agrobacterium-mediated in Planta transformation of maize germ cells].

Abstract-A transfer DNA (T-DNA) carrying the marker gene nptII was detected in the genomes of diploid and haploid maize plants obtained after the treatment of pistil filaments with a suspension of Agrobacterium during artificial pollination. PCR analysis of total DNA isolated from 155 canamycin-resistant diploid F1 seedlings revealed T-DNA insertions in the genomes of 111 plants (32.7% of the total number of analyzed seeds). The example of matroclinal haploids was used to demonstrate that T-DNA may be transported to the egg cell by the growing pollen tube (PT). Twelve out of 16 analyzed haploid plants contained the T-DNA insertion. The possible mechanism of the transfer of the Agrobacterium T-DNA to the maize genome during pollination is discussed.

[Effects of anesthetics on oxidative stress changes in patients with high anesthetic risk in the perioperative period of coronary bypass surgery].

The study included 44 patients who were divided into 3 groups according to the type of anesthesia. In group 1 patients (n = 15), initial anesthesia was accomplished by inhaled sevoflurane and intravenous fentanyl (2.5-3.5 microg/kg); basal anesthesia was performed with sevoflurane. In Group 2, midazolam, 0.1-0.15 mg/kg, fentanyl, 5.2 +/- 0.01 vg/kg, and ketamine, 0.85 +/- 0.13 mg/kg were given for induction. Basic anesthesia was carried out, by administering fentanyl in a dose of 4.71 +/- 0.4 microg/kg/hour, halothane, 0.5-1.5 ob %. In Group 3, midazolam, 1.2 +/- 0.01 mg/kg) and fentanyl, 7.8 +/- 0.6 microg/kg) were used to induce anesthesia. Basic anesthesia was effected with fentanyl, 5.31 +/- 0.5 microg/kg/hour, ketamine, and diprivan. Anesthetic management using halogen-containing inhalational anesthetics at coronary bypass surgery in patients at high anesthetic risk was ascertained to cause a significant reduction in the degree of manifestations of oxidative stress and facilitated a better intraoperative period. Sevorane was found to have the most significant effect on oxidative stress.

[In planta transformation of maize through inoculation of Agrobacterium into the silks].

Integration of T-DNA into the maize genome as a result of treatment of silks with Agrobacterium cells, containing activated vir genes, was demonstrated. In planta treatment of maize (Zea mays L) was performed during flowering in field. Cell suspension of Agrobacterium tumefaiciens strain GV3101(pTd33), carrying activated vir genes, was applied onto the previously isolated silks, which were afterwards pollinated with the pollen of the same cultivar. Integration of T-DNA into maize genome was confirmed by PCR (the nptII and gus reporter genes) and hystochemical staining of the seedling tissues, obtained from the transformed seeds. Amplification of the nptII gene showed the presence of about 60.3% of PCR-positive plants out of the total number of kanamycin-resistant seedlings examined, or 6.8% of the total of number of seedlings.

[Dependence of antimicrobial effect of micelle-capsulated therapeutics on the micelle size].

With the method of dynamic light scattering it was shown that the average size of micelles in the series of formulations based on various clindamycin salts, i. e. ClindHCl+Tween-20, ClindBz+Tween-20, ClindHCl+Cremafor-EL and ClindBz+Cremafor-EL increased from 6 to 20 nm. Investigations with the agar diffusion method revealed that the bactericidic action of the micelle-capsulated therapeutics did not depend on the micelle size within 6 to 20 mn. The concentration of the micellar clindamycin or gentamicin equal to 0.05 mcg/ml was bacteriostatic with respect to Micrococcus (Sarsina) luteus.

[Production of miniantibodies to Agrobacterium tumefaciens VirE2 virulence protein by the method of phage display].

The scFv miniantibodies to the recombinant protein VirE2 from Agrobacterium tumefaciens were obtained by the method of phage display. The miniantibodies were purified and tested using timmunodot method for binding to a recombinant protein from Escherichia coli and to the native protein VirE2 from A. tumefaciens. The functional activity of the miniantibodies was comparable to the activity of mouse polyclonal antibodies against the VirE2 protein.

[Cardiac arrhythmias during coronary bypass surgery without extracorporeal circulation].

Ventricular arrhythmias are recorded in most patients at coronary bypass surgery without extracorporeal circulation. The stages, such as the pulling of the sternal edges apart, the opening of the pericardium, the revision and dislocation of the heart, and revascularization of coronary arteries, are most dangerous due to the fact that they may lead to the development of arrhythmias. The major proarrhythmogenic factors at coronary bypass surgery without extracorporeal circulation are mechanical irritation of reflexogenic areas and myocardial ischemia, the mechanical factors playing the leading role in the development of ventricular arrhythmias.

[Isolation, purification, and identification of virulence protein VirE2 from Agrobacterium tumefaciens].

Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid, T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein with six histidine residues at the N-end was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2 which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.

[Leptospirosis in the Republic of Mordovia]. / Kharakteristika leptospirozov v Respublike Mordoviia.

Over 45 years, anthropurgic foci of Leptospira infection have been differently active in the Republic of Mordovia; in its population, the incidence of leptospiroses being greater than the average federal rates. The peak incidence of leptospirosis was observed at 10-11-year intervals: 1962-1963, 1971-1972, 1983, 1994-1995, 2001-2002 when outbreak and group morbidity were registered. There were 2.6- and 2.4-fold seasonal rises in morbidity in June to September with its peak in July-August, respectively. Cases of infection were notified in all the districts and towns of the republic, and in all landscape-environmental areas; group and outbreak incidence was found in 6 districts and environs of the town of Saransk in 1997-2002. Males aged 20-49 years are a group of risk for Leptospira infection; its incidence rates in age groups 0-14 and above 60 years are less than the average republican ones. Children (aged 0-14 years) and adolescents (aged 15-19 years) actively participate and determine outbreak and group mortality rates. Infection occurs most frequently while bathing in open water reservoirs, while using water from other water springs, going to the forest to pick mushrooms, berries, etc., while being engaged in laying in hay, fodder, and other feeds for animals. During registration of infection in Mordovia, the etiological pattern of human leptospirosis constantly changed, in 1992-2002 there were prevalent diseases caused by Grippotyphosa (60.96%), Canicola (7.12%), Seiroe (6.73%), Australis (6.54%), and Cynopteri (5.96%), in 1997-2002, the proportion of Australis (11.2%) increased and that of Canicola (4.7%) decreased. The leptospira serogroups Icterohaemorrhagiae, Tarassovi, Seiroe were prevalent animals. There was no correlation of the etiological environment of leptospiras isolated from agricultural animals and human beings in 1997-2002, which proves the leading role of natural leptospirosis foci and rodents as a source of infection.

[The system of neurohumoral regulation during bypass surgery without extracorporeal blood circulation]. / Sostoianie sistemy neirogumoral'noi reguliatsii organizma vo vremia operatsii koronarnogo shuntirovaniia bez iskusstvennogo krovoobrashcheniia.

The activity of sympathyco-adrenal, and of serotonin-hystain-cholinergic systems was studied in 34 patients operated for the ischemic heart disease without extracorporeal blood circulation. A moderate increase of parameters characterizing the condition of sympathyco-adrenal, and of serotonin-hystain-cholinergic systems was noted in patients in the perioperative period. The activity of the studied systems reduced due to a total anesthetization. At the same time, the activation of the factors of the neurohumoral system was limited by the adaptation norms with sufficient reserves being preserved.

[Formation of TRA-dependent surface structures in Agrobacterium tumefaciens and their absence in R1 (deltatraR) mutant]. / Obrazovanie TRA-zavisimykh poverkhnostnykh struktur u Agrobacterium tumefaciens i ikh otsutstvie u mutanta R1 (deltatraR).

Agrobacteria have Ti plasmid DNA delivering systems for the transfer to recipient cells by the conjugation mechanism. This transfer is absolutely dependent on induction tra genes. It is not clear which tra-dependent surface (extracellular) proteins (structures) are involved in the transport mechanism and whether these proteins also play a role in the contact formation. SDS-PAGE electrophoresis of proteins released from the cell showed disappearance of 63 and 67 kD proteins in R1(delta traR) strain, which were found in the growth medium and triton extract from the outer membrane of Ti plasmid-harboring A. tumefaciens R10 strains. The traR defective mutant did not express these proteins and had a higher hemagglutination and flocculation capacity than the wild strain. On the other hand, the wild strain showed D-galactose and N-acetyl-galactosamine specific hemagglutination which was not shown by traR mutant. Motility and chemotactic behavior of traR mutant in semisolid medium were defective. As a rule, one (or rarely two) thread-like connections in vir(-) and tra(+) conditions were observed on the agrobacterial cell surface. SDS pretreatment of agrobacterial cells had a significant effect on the expression of tra-dependent surface structures.

[Study of extracellular structures of Agrobacterium involved in bacterial and plant interactions]. / Izuchenie vnekletochnykh struktur agrobakterii, uchastvuiushchikh v kontaktakh s bakterial'noi i rastitel'noi kletkami.

Agrobacterial cells produced straight microfibrils not only when in contact with wheat seedling roots, but also when in contact with each other. After 2 h of incubation, agrobacterial cells were found to form aggregates, in which the cells were in contact either directly or through thick straight microfibrils (bridges) of an unknown composition. The majority of the microfibrils were susceptible to attack by cellulase, although some of them showed resistance to this enzyme. Like the wild-type flagellated agrobacteria, their bald mutants produced long straight microfibrils. The cells surface structures of agrobacteria were examined by labeling them immunocytochemically with colloidal gold conjugated antibodies against O-specific lipopolysaccharides, Vir proteins, and cellulase. Agrobacterial cells treated with acetosyringone and brought into contact were found to contain subpolar and polar cell surface structures. Antibodies against the VirB2 protein were able to interact with a tuft of thin microfibrils located on one pole of the agrobacterial cell, whose vir genes were induced by acetosyringone, but were unable to interact with the surface structures of the agrobacterial cells aggregated in liquid medium in the absence of wheat seedlings.

[Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes]. / Perenos T-DNK iz agrobakterii v rastitel'nuiu kletku cherez kletochnye stenki i membrany

Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma. Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E. coli, A. tumefaciens) to recipient (bacterial or plant) cells. Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes. Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells. Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer. Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins.