Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries

The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring.     

Deposition of callose in treatment of cells of the tomato plant (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.) with biotic elicitors

The dynamics of induced accumulation of callose in tomato cells is studied. Localization of callose in the cells is studied by methods of fluorescence microscopy and the time needed for quantitative determination of callosa is optimized. The quantity of callose in tomato cells treated with different biotic elicitors is established. A nonlinear dependence of the deposition of callose on the concentration of chitin oligomers containing 3–5 fragments of N-acetylglucosamine is established and a proportional increase in the content of callose in the cells with increasing concentration of chitin dimer and chitosan in the culture medium is demonstrated.     

Callose accumulation during treatment of tomato (Lycopersicon esculentum L.) cells with biotic elicitors

Time-course of induced accumulation of callose in tomato cells has been studied. Localization of callose in L. esculenthum cells was investigated by fluorescent microscopy technique, and the optimal time for its determination was found. Callose accumulation in tomato cells treated with different biotic elicitors was determined. Nonlinear dependence between callose accumulation and concentration of chitin oligomers (with 3-5 N-acetylglucosamine fragments) was established. Increasing of callose accumulation in tomato cells was proportional to the increase of concentration ofchitin dimer and chitosan in the culture medium.     

Interaction among proteins and peptide libraries in proteome analysis: pH involvement for a larger capture of species

When capturing proteins via combinatorial peptide ligand libraries, a method well known for drastically reducing the concentration of high-abundance proteins and substantially magnifying the signal of low-abundance species, thus leading to the discovery of a large number of proteins previously undetected in proteomes, we had constantly noticed that there would be a loss of species initially present in the untreated sample, to the tune of 5%, up to 15% in some cases. Such losses are a nuisance and hamper to some extent the unique performance of the method. In order to verify if such losses could be reduced and also to understand some mechanisms of the capture process, we introduce here an important variant to the capture operation, up to the present carried out in physiological saline at pH 7.2. In this novel protocol, the binding step is conducted at three different pH values, namely the standard one at pH 7.2, plus two additional processes, at acidic (pH 4.0) and alkaline (pH 9.3) pH values. Indeed the capture process is more extensive, with a number of additional species captured at the two pH extremes in sera and other proteomes. Interestingly, at pH 4.0 newly detected proteins were mostly acidic, while at the alkaline pH additional protein species were more evenly distributed throughout the pI range towards the alkaline area. The role of pH in the complex mechanism of binding among the hexapeptide library and the various proteomes being analyzed is discussed and evaluated. Due to significant changes in protein patterns with pH, recommendations are thus made to increase the possibility to find additional gene products illustrated by two examples (snake venom and leaf protein extract). Keeping under control the environmental pH when facing reproducibility studies or for comparative proteomics profiling is also a general rule suggested by this study.     

Analysis of polymorphism at nine nuclear genome DNA loci in maris

Population genetic survey of the indigenous populations of the Marii El Republic, represented by the two major ethnographic groups of Maris, Meadow (five samples from Morkinsk, Orshansk, Semursk, Sovetsk, and Zvenigovsk districts) and Mountain (one sample from Gornomariisk district) Maris, was carried out. All Mari groups were examined at nine polymorphic DNA loci of nuclear genome, VNTR(PAH) (N = 422), STR(PAH) (N = 152), VNTR(ApoB) (N= 294), VNTR(DAT1) (N = 363), VNTR(eNOS) (N = 373), ACE (N = 412), IVS6aGATT (N = 513), D7S23(KM.19) (N = 494), and D7S8 (N = 366). Allele and genotype frequency distribution patterns were obtained for individual samples and ethnographic groups, as well as for the ethnic group overall. In each of six Mari samples examined, the deficit of heterozygotes was observed, i.e., the mean observed heterozygosity was lower than the expected one. The indices of mean heterozygosity, Hs = 0.455, and interpopulation differentiation, FST = 0.0024, for the Mari gene pool were obtained using a set of DNA markers analyzed. Analysis of the genetic distances and between population differentiation (FST) showed that the main part of genetic diversity in Maris was determined by the differentiation between the populations of Meadow Maris. The contribution of the differences between the ethnographic groups of Mountain and Meadow Maris to the ethnic gene pool was small. It is suggested that the main role in the formation of the Mari gene pool is played by the geographic factor.     

pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries

The pre-treatment of biological extracts with the aim of detecting very low-abundance proteins generates complexity requiring a proper fractionation. Therefore the success of identifying all newly detectable species depends on the selected fractionation methods.In this context and starting from a human serum, where the dynamic concentration range was reduced by means of a preliminary treatment with a combinatorial hexapeptide ligand library, we fractionated the sample using a novel method based on the differences in isoelectric points of proteins by means of Solid-State Buffers (SSB) associated with cation exchangers. The number of fractions was limited to four and was compared to a classical anion exchange method generating the same number of fractions.What was observed is that when using SSB technology the protein redundancy between fractions was significantly reduced compared to ion exchange fractionation allowing thus a better detection of novel species. The analysis of trypsinized protein fractions by nanoLC-MS/MS confirmed that the SSB technology used is more discriminant than anion exchange chromatography fractionation.A sample fractionation by SSB after the reduction of dynamic concentration range can be accomplished without either adjustment of pH and ionic strength or protein concentration and cleanup. Both advantages over either classical chromatography or isoelectric fractionations allow approaching the discovery of markers of interest under easier conditions applicable in a variety of fields of investigation.     

Seasonal distribution and fatty acid composition of littoral microalgae in the Yenisei River

We studied fatty acid (FA) composition of littoral microalgae in the fast-flowing oligotrophic river, the Yenisei, Siberia, monthly for 3 years. Seasonal dynamics of species composition had similar patterns in all the studied years. In springs, a pronounced dominance of filamentous green algae occurred, in summer and autumn diatoms were abundant, and in late autumn and winter epilithic biofilms consisted primarily of cyanobacteria and detritus. In general, FA composition of the algal periphytic community was dominated by 16:0, 16:1ω7, 20:5ω3, 14:0, and 18:3ω3 throughout the studied period. Several groups of FAs, which had peculiar seasonal dynamics, were differentiated by statistical analysis based on a method of correlation graphs. The seasonal changes in FA composition could be partly explained by the seasonal succession of species composition of the community. Besides, we found that populations of both diatom and green algae grown in summer at a higher water temperature were lower in polyunsaturated fatty acids than those in spring, at a lower temperature. Hence, we suppose that the regular seasonal dynamics of FA composition of the studied littoral microalgae was driven both by changes in species composition and by temperature adaptations of the algal populations. The highest content of essential polyunsaturated FAs, eicosapentaenoic and docosahexaenoic acids, in the spring “psychrophilic” populations of diatoms could make them of the higher nutritive value for zoobenthic primary consumers.