Protein Reabsorption in the Amphibian Kidney: Comparative and Evolutionary Aspects

Protein reabsorption in the renal proximal tubule (PT) is a vitally important process which prevents the loss of filtered proteins and provides their participation in subsequent metabolism. Despite considerable changes in renal structure and function in the process of evolution, very little is known about the functional similarities or specifics of tubular protein reabsorption in the kidney of lower vertebrates compared with the mammalian and human kidney. This article presents an overview of our recent studies on protein reabsorption in the kidney of amphibians, which are used as one of the main animal models for current biological and biomedical research. In frogs, newts, and rats, the absorption capacity of epithelial PT cells was studied after the introduction of green fluorescent protein (GFP), yellow fluorescent protein (YFP), and lysozyme. Molecular mechanisms of receptor-mediated protein endocytosis were also investigated by immunohisto- and immunocytochemistry, electron, fluorescent, and laser scanning confocal microscopy

Dimensions of social capital

Objective to describe the manifestations of social capital in the context of the main factors affecting its formation which will enable to systematize the existing ideas about the dimensions of social capital. Methods abstractlogical and dialectical phenomenological methods. Results social capital is an important concept in the economic system but it is difficult to identify and measure. The article analyzes the phenomenon of social capital as well as approaches to its definition. Based on the literature analysis five sections are identified in which the concept of social capital is characterized. It is shown that the institutional environment is the basis for the formation of social capital. It is revealed that the individual qualities of a personality can both contribute emotional intelligence etc. and prevent closeness for communication inability to listen conflictgenerating character etc. the accumulation of social capital. The content of the concept of family social capital is shown. The predominant influence of social capital on the development of small firms in comparison with medium and large organizations is revealed. The influence of civic activity on economic development through social capital is studied. Scientific novelty five dimensions of social capital are identified 1 social capital and individual qualities of personality 2 social capital and family ties 3 social capital and corporate culture 4 social capital and civic activity 5 social capital and institutional environment. It is determined how these patterns affect economic development. Practical significance the understanding of the main dimensions of social capital revealed in the work makes it urgent to identify the structure of social capital and creates a basis for further research in this area

Comparison of the Formation of Plant–Microbial Interface in <i>Pisum sativum</i> L. and <i>Medicago truncatula</i> Gaertn. Nitrogen-Fixing Nodules

Different components of the symbiotic interface play an important role in providing positional information during rhizobial infection and nodule development: successive changes in cell morphology correspond to subsequent changes in the molecular architecture of the apoplast and the associated surface structures. The localisation and distribution of pectins, xyloglucans, and cell wall proteins in symbiotic nodules of Pisum sativum and Medicago truncatula were studied using immunofluorescence and immunogold analysis in wild-type and ineffective mutant nodules. As a result, the ontogenetic changes in the symbiotic interface in the nodules of both species were described. Some differences in the patterns of distribution of cell wall polysaccharides and proteins between wild-type and mutant nodules can be explained by the activation of defence reaction or premature senescence in mutants. The absence of fucosylated xyloglucan in the cell walls in the P. sativum nodules, as well as its predominant accumulation in the cell walls of uninfected cells in the M. truncatula nodules, and the presence of the rhamnogalacturonan I (unbranched) backbone in meristematic cells in P. sativum can be attributed to the most striking species-specific features of the symbiotic interface

List of primers used for PCR-amplification of full-size genes encoding the studied proteins.

a<p>restriction enzyme sites are underlined in the primer sequences.</p>b<p>to express hexokinase in <i>Pichia pastoris</i>, the forward primer with the <i>Bgl</i>II site instead of <i>Xho</i>I was used for PCR amplification of gene copies.</p

Heterologous expression of <i>A. locustae</i> proteins in yeast <i>P. pastoris</i> cells.

<p>A. Expression of microsporidian proteins without any exogenous signal peptides in yeast <i>P. pastoris</i> led to the secretion of two LRR-proteins, hexokinase and trehalase. Lane 1, yeast cells before methanol induction; lane 2, cells expressing the studied protein after adding methanol; lane 3, culture medium concentrated approximately 50 times; lane 4, yeast cells expressing another parasite protein after methanol induction (control cells); lane 5, concentrated medium after cultivation of cells expressing another parasite protein (control medium). Samples were analyzed by immunoblotting with Abs specific to the expressed protein. B. Though α/β-hydrolase was not found in the culture medium, a significant amount of enzyme accumulated in the insoluble fraction of <i>P. pastoris</i> cell homogenate. Yeast cells were broken in the presence of 0.3 M sucrose and homogenate was cleared at 270 <i>g</i> for 4 minutes and then centrifuged at 18,000 <i>g</i> for 20 minutes. Western blot analysis of the supernatant (lane “sup”) and pellet (lane “pel”) showed precipitation of the enzyme, which suggests its association with cell membranes.</p

Analysis of ORFs encoding <i>Antonospora locustae</i> proteins.

<p>A. ClustalW alignment demonstrated that most genes (ORFs) encoding LRR-proteins in <i>A. locustae</i> genome form two large families. The location of the new LRR-protein of family A found in the microsporidian genome and expressed in this study is marked with two asterisks. The position of the protein encoded by ORF 515 of family B is indicated by one asterisk. Proteins with secretion signal peptides predicted by SignalP server are indicated by a plus sign. B. The amino acid sequence of the novel <i>A. locustae</i> LRR-protein of family A. C. Compared to NCBI data, two extra C-terminal repeats VPENPLVSTLSVP(E/D)DLP(A/T)CTQH were found in cloned α/β-hydrolase.</p

Western blot assay of <i>A. locustae</i> proteins.

<p>Samples of <i>A. locustae</i> intracellular stages isolated in Percoll density gradient (lane St), infected host cytoplasm obtained by gentle homogenization of locust fat bodies following sedimentation of parasites (lane M), and cytoplasm of uninfected (control) fat bodies prepared in the same manner (lane C) were equalized in protein concentration. The proteins transferred on nitrocellulose membranes were stained either by Ponceau S or by Abs against <i>A. locustae</i> Hsp70, hexokinase, α/β-hydrolase, two LRR-proteins, and trehalase. This experiment identified parasite proteins in the infected host cytoplasm.</p

Immunolocalization of <i>A. locustae</i> hexokinase in infected cells.

<p>A. The presence of microsporidial protein in infected cytoplasm and its accumulation in host nuclei was demonstrated using wide field immunofluoresence microscopy. The top row demonstrates accumulation of hexokinase in three nuclei of infected host cells. A nucleus of an uninfected cell is located in the bottom left corner. The bottom row shows accumulation of hexokinase in the single nucleus of an infected cell surrounded by a number of uninfected ones. The thick and thin arrows on the DAPI images indicate nuclei of infected and uninfected host cells, respectively. The nucleus of infected cells on the periphery of the invasion region (top row) is marked by the middle arrow. Scale bars, 10 μm. B. Colocalization of <i>A. locustae</i> hexokinase and Hsp70 were analyzed by confocal microscopy. Scale bars, 5 μm.</p

Construction of scFv Antibodies against the Outer Loops of the Microsporidium <i>Nosema bombycis</i> ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or “camelization” of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs