Protein glycosylation in the spores of the microsporidia Paranosema (Antonospora) grylli

Long adaptation of microsporidia, a large group of fungi-related protozoa, to intracellular lifestyle has resulted in drastic minimization of a parasite cell. Thus, diversity of carbohydrates in microsporidia glycoproteins and proteoglycans is expected to be restricted by O-linked manno-oligosaccharides because three genes involved in O-mannosylation of proteins and no components of N-linked glycosylation machinery were found in genome of human pathogen Encephalitozoon cuniculi. In this study we investigated glycosylation of spore proteins of microsporidia Paranosema (Antonospora) grylli infecting crickets Gryllus bimaculatus. Using periodic acid-Shiff reagent staining we have demonstrated that some P. grylli spore proteins are highly-glycosylated. The major polar tube protein (PTP1) of 56 kDa was shown as the most intensively decorated band. The experiments with N-glycosidase F and WGA lectin did not reveal any N-glycosylated proteins in P. grylli spores. At the same time, incubation of major spore wall protein of 40 kDa (p40) with mannose specific lectin GNA resulted in specific binding that was reduced by pretreatment of the protein with mannosidases. Interestingly, in spite of PTP1 glycosylation, polar tube proteins extracted from P. grylli spores were not precipitated by GNA-agarose. Since P. grylli and E. cuniculi are distantly related, our data suggest that dramatic reduction of protein glycosylation machinery is a common feature of microsporidia.     

An Artificial Protein with the Biological Activity of the Differentiation Factor for the HL-60 Cell Line of Human Promyelocyte Leukemia

The ABB-df artificial protein was prepared by inserting the TGENHR biologically active peptide corresponding to the 41–46 sequence of the differentiation factor for the HL-60 cell line of the human promyelocyte leukemia into the N-terminus of the polypeptide chain of albebetin, an artificial protein with the preset structure. The ABB-df protein was found to induce the differentiation of HL-60 cells and to inhibit their proliferation; its efficiency was almost the same as that of the starting peptide. According to CD spectroscopy, the inclusion of the peptide fragment into albebetin exerts virtually no effect on the regular secondary structure of albebetin.     

Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

Features of compensatory-adaptive reactions of an organism in female representatives of the Evenk ethnos

Adolescent girls and women of reproductive age, female representatives of the Evenk ethnos and Europeoids that co-inhabit the north of the Irkutsk oblast, were examined. Changes in metabolic reactions in the small indigenous ethnic group were found to have a more adaptive character than in the Europeoids. In the Evenk vs. Europeoid girls, an increased activity both of glucocorticoid function of the adrenal glands and non-enzymatic antioxidant defense system was detected. In spite of the higher total cholesterol (TCh) level in women vs. girls in both ethnic groups, in the Evenk group TCh tended to decline. Changes in the antioxidant defense system of the Evenk women manifested themselves as an increased total antioxidant activity of blood vs. Europeoids as well as higher TCh and lower retinol levels vs. adolescent girls.     

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.     

The effect of electroacupuncture on the neuronal responses of the oral trigeminal nucleus in the cat during nociceptive and non-nociceptive stimulation

Effects of electroacupuncture (EAP) on the responses of different functional types of neurons of the oral trigeminal nucleus (OTN) by nociceptive and non-nociceptive stimulation were studied in acute experiments on adult cats. It was demonstrated that the main part of neurons of the OTN is a wide dynamic range of neurons. Characteristic feature of the OTN is neurons with low-threshold pulp afferent input. EAP inhibit nociceptive responses of neurons (preferentially nonspecific neurons), while responses to non-nociceptive stimulation are not changed at all. The results are discussed from the point of view that OTN takes part in nociceptive and non-nociceptive reactions.     

Development and characterization of antibodies against aflatoxins

A panel of ten monoclonal antibodies against aflatoxins B1, B2, and G2 was produced and comprehensively characterized. The affinity and cross reactivity of these antibodies were determined using the methods of direct, indirect, and competitive ELISA. The structures of monoclonal antibody genes were comprehensively studied and the variable and constant regions of the antibody genes were cloned and sequenced. Sequencing analysis confirmed the results of isotyping the light and heavy antibody chains obtained by ELISA. Variable and constant fragments of the antibody genes were cloned into a bicistron expression vector for the recombinant Fab-fragment for one of the antibodies expressed in Escherichia coli and purified. Thus, data were obtained that can be useful for the development of an aflatoxin detection system on the basis of the described monoclonal antibodies and the creation of recombinant antibodies with changed parameters of specificity using protein engineering methods.     

Antiarrhythmic activity of 4,6-di(het)aryl-5-nitro-3,4-dihydropyrimidin-(1H)-2-ones and its effects on arterial pressure in rats

The antiarrhythmic activity of 4,6-di(het)aryl-5-nitro-3,4-dihydropyrimidin-(1H)-2-ones toward two types of experimental rat arrhythmia has been studied. With CaCl(2) induced arrhythmia model, several agents have demonstrated high antiarrhythmic activity and the lack of influence on arterial pressure of rats.     

Metabolic integration of organisms within symbiotic systems

Adaptation of organisms to coexisence in symbiotic systems is usually related to significant metabolic changes resulting in the integration of the biochemical pathways of the partners. In the symbioses between plants and nitrogen-fixing organisms, between heterotrophic and autotrophic organisms, as well as between animals and microorganisms providing the consumption of plant biomass, the systems of C- and N-metabolism, controlling the utilization of various sources of nitrogen (N2, organic and inorganic compounds, metabolic waste of the host) and carbon (CO2, plant polymers), of the partners are tightly integrated. Bilateral biochemical links between partners are typical to mutualistic symbioses (wherein biotrophic nutrition predominates, in some cases including necrotrophy of secondary origin). In antagonistic symbioses, unilateral links predominate, though active assimilation of the pathogen's secondary metabolites by the host is also possible. In most mutualistic symbioses, integrated metabolic ties have derived from trophic chains in biocenoses (syntrophic consortia, "predator-prey" systems), but not from the systems where the pathogens consume host metabolites. At the same time, molecular analysis of symbiotic interactions has shown that symbioses considerably differ from biocenoses, where the cycling of nutrients and energy implies no functional integration of the partner's genes.