Study of total activity of natural lipid antioxidants by chemiluminescence method

The total antiradical activity of lipid antioxidants extracted from organs and tissues of fish Coregonus peled (Gmelin) was investigated using chemiluminescence method. It has been established that lipids contain antioxidants of two types. The bioantioxidants of the first type have a comparatively high efficiency constant K7eff. = (2.4-3.2) 10(6) M-1.s-1, whose value is 100 times more than that of the constant of the second type inhibitors K7eff. = (3.5-5.0) 10(4) M-1.s-1. Using the method of thin-layer chromatography such individual antioxidants of lipids as tocopherol, ubiquinon, ubichromenol were separated and quantitatively studied, as well as recorded in the presence of vitamin K, A, cholesterol. It is shown that the quantitative content of high-activity antioxidants in lipid of different kinds substantially varies (0.5-17.1) 10(-4) M; the low level of their content has been recorded for internal fat and brain lipids, the high one--for the lipids present in immature eggs, red muscles and liver.     

The enzyme-antienzyme interactions of a number of Burkholderia pseudomallei hydrolases with monoclonal immunoglobulins

Phosphatase, phospholipase C and a proteolytic complex, including casein- and hemoglobin-hydrolases, have been isolated from cell-free extracts of B. pseudomallei cultivation medium. A set of monoclonal antibodies (McAb) to the antigenic complexes of this infective agent has been obtained. Conditions for the study of the interaction of the enzymes and McAb have been worked out, and most of the 14 immunoglobulins under study have been shown to neutralize 2-3 hydrolases each. Three types of McAb, selectively inhibiting only the proteolytic complex, only casein-hydrolase and only phospholipase C, have been identified.     

The isolation of monoclonal antibodies to Cryptococcus neoformans antigens

In this work conditions for the reproduction of hybridoma technology, specially adapted to C. neoformans, for obtaining monoclonal hybridomas (McAb) to diagnostically significant antigens of C. neoformans, the infective agent of cryptococcosis, are presented. The advantages of using the short-time cycle of stimulation of mouse B lymphocytes with low doses of C. neoformans capsular polysaccharide and the effectiveness of the hybridization of mouse spleen cells with myeloma cells, line Sp2/0, are shown. Four lines of stable hybridomas, producing McAb to different epitopes of C. neoformans surface antigens, have been obtained. The specific activity of McAb has been studied in the indirect immunofluorescence assay, the cytochemical and solid-phase enzyme immunoassays (EIA). McAb 3E2, Cr2 and 2G9 have been shown to be suitable for use in diagnostic EIA systems.     

Antigen 8 biosynthesis during cultivation of Burkholderia pseudomallei and B. mallei

The dynamics of the antigen 8 synthesis in Burkholderia pseudomallei and B. mallei under conditions of their submerged was studied. Differences in the intensity of this antigen synthesis in two pathogenic Burkholderia species were established and the producer strains, most effective with respect to this sign, were selected.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Use of infusoria Paramecium caudatum for the evaluation of the toxicity of antigens of different causative quarantine agents

The results of the evaluation of the toxicity of bacterial antigens obtained from the causative agents of plaque, glanders, melioidosis, cholera on infusoria of the species P. caudatum, as well as on cell lines L-929, CHO K-1 and peritoneal macrophages of BALB/c mice, are presented. As revealed in this study, the method of toxicity determination on infusoria is similar in its sensitivity to the methods of testing on. CHO K-1 and L-929 cells, but the former is simpler, more available and permits the determination of toxic doses producing disturbances in the vital activity of the infusoria, but not leading to their death.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

The determination of the location of the antigens of the causative agent of melioidosis at the ultrastructural level by using monoclonal antibodies]

The indirect immunoperoxidase method with the use of monoclonal antibodies has been employed to determine the localization of antigens of melioidosis agents. The formed reaction products were then detected by the electron microscopic analysis. The most important in melioidosis pathogenesis antigens 6 and 8 (AG 6 and AG 8) were determined to localize on the cell wall and in the capsule-like mucous layer, respectively. Monoclonal antibodies to AG 8 due to their marked cross-linking properties promote stabilization, preservation and detection of labile melioidosis agent. At the same time the mucous layer prevents monoclonal antibodies to AG 6 to access to the corresponding antigen, localized on the cell wall and makes its detection difficult.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).