Sergey F. Beresten

Enteric alpha defensins in norm and pathology

Microbes living in the mammalian gut exist in constant contact with immunity system that prevents infection and maintains homeostasis. Enteric alpha defensins play an important role in regulation of bacterial colonization of the gut, as well as in activation of pro- and anti-inflammatory responses of the adaptive immune system cells in lamina propria. This review summarizes currently available data on functions of mammalian enteric alpha defensins in the immune defense and changes in their secretion in intestinal inflammatory diseases and cancer.

Molecular and cellular studies of tryptophanyl-tRNA synthetases using monoclonal antibodies Remarkable variations in the content of tryptophanyl-tRNA synthetase in the pancreas of different mammals

The content of Trp-tRNA synthetase in pancreas and liver of cattle, sheep, swine, rat, rabbit and man was assayed by direct radioimmunoblotting with a 125I-labelled monoclonal antibody Am1, specifically interacting with any eukaryotic Trp-tRNA synthetase. Its content in the organs studied, with the exception of bovine and sheep pancreas, was found to be 0.002-0.012% of total proteins. The enzyme content in bovine pancreas was about 0.2% of total proteins, i.e. 70 times higher than in bovine liver; similar correlations were found for sheep. The Trp-tRNA synthetase levels in each organ varied from animal to animal of the same species by not more than a factor of four; these individual variations cannot affect the conclusion about the profound differences in the levels of the enzyme in pancreases of Ruminantia and of the other mammalians. As shown by indirect immunofluorescence technique, bovine Trp-tRNA synthetase is mainly located in the exocrine part of the pancreas. Moreover, the immunoreactive material is detectable also in bovine (not human) pancreatic juice. The abnormally high Trp-tRNA synthetase content in the ruminant pancreas may be connected with unknown function(s) of this protein somehow related to the peculiarities of digestion of these mammals.

Immuno-chemical non-cross-reactivity between eukaryotic and prokaryotic seryl-tRNA synthetases

Monospecific polyclonal antibodies (pAbs) against highly purified bovine seryl‐tRNA synthetase (SerRS, EC 6.1.1.1) were prepared and their specificity tested. The interactions of pAbs with SerRS from different organisms were investigated by protein immunoblotting and ELISA methods. pAbs inhibit eukaryotic SerRS aminoacylating activity and exert no effect on SerRS activity from prokaryotes. It is proposed that prokaryotic and eukaryotic SerRS evolve from different ancestor genes.

Role of exposed cytosine residues in aminoacylation activity of tRNATrp.

Selective chemical modification of tRNA is a conventional procedure used to detect bases which are essential for the interaction of tRNA molecules with synthetases (see [l-3]). Bisulfite is a suitable reagent in such studies since it permits conversion of cytosine residues in an RNA molecule into uridine residues (see [4]). This reaction has been employed in studying a number of t RNA-synthetase complexes [l-S]. Here, we have detected cytosine residues in bovine tRNATv whose conversion into uracil residues makes the tRNA lose its ability to be aminoacylated with tryptophan in the presence of homologous tryptophanyl-tRNA synthetase. The conversion of two cytosine residues in the anticodon loop (C3, and C33) inactivates the tRNA whereas the C + U conversion of the second base in the anticodon (C,), as well a similar conversion of cytidines at the CCA-terminus of the molecule, does not inactivate tRNA. A possible effect produced by modifying the anticodon loop on anticodon stem structure is discussed. Fine Chemicals); [Y-~~P] ATP, spec. act. 3000 Ci/mmol (Radiochemical Centre, Amersham); sodium bisulfite (granular) (Mallincrodt Chemical Works). tRNAT’P (8 A 260 units) was modified with bisulfite in 0.15 ml 1.5 M Na2S205 (PH 5 .S) containing 10 mM MgC12 at 20°C for 2 h. tRNATv lost 50% of its activity within this time. The reaction was stopped by gel filtration of the solution on a column packed with Sephadex G-50 (fine). The uridine bisulfite adducts were demodified by incubating tRNA in 0.2 ml 0.1 M Tris-HCl (pH 9.0) for 24 h.

Bovine tryptophanyl-tRNA synthetase and glyceraldehyde-3-phosphate dehydrogenase form a complex

Bovine tryptophanyl-tRNA synthetase is able to form a complex with glyceraldehyde-3-phosphate dehydrogenase. The complex formation (i) does not influence the tryptophan-dependent PPi-ATP exchange reaction and (ii) involves predominantly the N-terminal dispensable domain of the synthetase. Glyceraldehyde-3-phosphate dehydrogenase was shown to be capable of interacting simultaneously with tryptophanyl-tRNA synthetase and with ribosomal RNA to form a ternary complex. It is proposed that compartmentation of some aminoacyl-tRNA synthetases in certain cases might be achieved via adapter molecules which can bind at once to ribonucleic acids and to aminoacyl-tRNA synthetases.

A rapid method for mapping exposed cytosines in polyribonucleotides. Application to tRNATrp (yeast, beef liver).

A rapid method for mapping exposed cytosine residues in 5′-[32P]-labeled RNA molecules is suggested. The exposed cytosines (Cs) are converted into uracyls (Us) by bisulphite treatment at pH 5.8 in the presence of Mg2+, followed by complete modification of the residual (non-exposed) Cs by a methoxyamine and bisulphite mixture at pH 5.0. The control RNA is modified only by methoxyamine and bisulphite without the preliminary C→U conversion. The location of the exposed Cs is determined by comparing the products of partial T1, T2, A and U2 ribonuclease digestions of the C → U converted and control RNAs after slab gel polyacrylamide electrophoresis and autoradiography. The method has been applied for mapping exposed cytosine bases in tRNATrp (yeast) which have been found in the anticodon loop and at the 3′-end of the molecule. In tRNATrp (beef liver), in addition to the same exposed bases, C in the diHU-loop is exposed. The data obtained are in full agreement with that is known about exposed Cs for other tRNAs.

Random-splitting of tRNA transcripts as an approach for studying tRNA-protein interactions

Location of phosphodiester bonds essential for aminoacylation of bovine tRNTrp was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21, 22, 36–38, 57–59, 62 and 64 were critical for aminoacylation capacity of tRNATrp‐transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and in the regions presumably outside the contact area as well. These results indicate that for maintenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non‐modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed in this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.