A method of creation a cell monolayer based on organotypic culture for screening of physiologically active substances.

We developed a method of culturing and phenotyping of a monolayer of cells of the retinal tissue, thymus and spleen on the basis of organotypic culture. All characteristic types of neurons and fibroblasts were found in their microenvironment in the retinal cell monolayer. Lymphocytes, macrophages, and fibroblasts were verified in the monolayer of thymus and spleen cells. Histological staining, immunocytochemistry, and electron microscopy demonstrated the possibility of assessing the differentiation degree and functional activity of the cell monolayer. The developed technique preserves cell-cell interactions and a variety of cell types characteristic of the examined organ in the monolayer. This opens up new prospects for its application in basic research and in screening of different physiologically active substances.

Characterization of Northwest Africa 6286 and 7857 ordinary chondrites using X-ray diffraction, magnetization measurements and Mössbauer spectroscopy.

Northwest Africa (NWA) 6286 and 7857 meteorites were studied in detail by using optical microscopy, scanning electron microscopy with energy dispersion spectroscopy, X-ray diffraction, magnetization measurements and 57Fe Mössbauer spectroscopy with a high velocity resolution. The main and the minor iron-bearing phases were identified in both meteorites. The unit cell parameters as well as Fe2+ and Mg2+ cation distribution were determined for the M1 and M2 sites in silicate microcrystals. Saturation magnetic moments were obtained for both meteorites. Mössbauer parameters for the main and the minor iron-bearing microcrystals were estimated and compared for NWA 6286 and NWA 7857 LL6 ordinary chondrites. The Fe2+ and Mg2+ cation partitioning, distribution coefficient and temperature of cation equilibrium distribution were estimated for silicate microcrystals using X-ray diffraction and Mössbauer spectroscopy.

NMR studies of a complex of RNAse from Penicillium brevicompactum with dinucleoside phosphonate and the implications for the mechanism of enzyme action.

The chemical-shift dependences of the proton signals of the guanosine and uridine moieties were measured as a function of the relative amount of GpcU complexed with RNase Pb1 (EC 3.1.27.3). The equal values of the chemical-shift changes of the guanosine C8-protons on complex formation between GpcU and RNase Pb1 and that of the 3'-GMP and RNase Pb1 allow to conclude that the guanosine base is bound in the same manner in these protein-ligand complexes. The guanosine moiety of GpcU is also most probably bound in the syn-conformation. The absence of changes in both the linewidths and the chemical shifts of the C1', C5 and C6-proton signals of the uridine on complex formation indicates that the uridine moiety of the dinucleoside phosphonate is not immobilized in the complex. The pH dependences of the chemical shifts of the C2-protons of the histidine-imidazole ring of RNase Pb1 and that of the 31P of GpcU in the RNase complex were studied. The results suggest that there is a direct interaction between the phosphonate group of the ligand and the protonated imidazole ring of His-90. The side groups of His-38 and Glu-56 are hydrogen bonded to each other at neutral pH and they are located in the vicinity of the phosphonate group of GpcU. When the carboxyl group of Glu-56 is protonated the His-38 imidazole ring forms a new hydrogen bond with one of the phosphoryl oxygens of the phosphonate group. On the basis of these results we propose the mechanism of action of RNase Pb1 which is probably also true for RNase T1.

The effect of net charge on the solubility, activity, and stability of ribonuclease Sa.

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

Correlative variations of the free energies for enzyme-substrate complex formation and the transition-state stabilization for RNases.

It was found for RNases of different specificities that changes in the free energy for substrate-enzyme binding induced by variations in the nucleotide base structure are accompanied by proportional changes in kcat/Km. This was considered to be a consequence of the strain in the enzyme-substrate complex.

Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease.

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease.

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).

Stereoelectronic effects in RNase-catalysed reactions of dinucleoside phosphate cleavage.

The rate at which dinucleoside phosphates are cleaved by RNases is supposed to be determined by the mole fraction of enzyme-substrate complexes in which the phosphodiester moiety of a dinucleoside phosphate has a highly reactive conformation. The mole fraction of such complexes for a particular RNase depends on the nature of a nucleoside at the O5'-end of the phosphodiester bond. Experimental data are presented to support this hypothesis.

Thermostability of the barnase-barstar complex.

Scanning microcalorimetry was used to study heat denaturation of barnase in complex with its intracellular inhibitor barstar. The heat denaturation of the barnase-barstar complex is well approximately by two two-state transitions with the lower temperature transition corresponding to barstar denaturation and the higher temperature one to barnase denaturation. The temperature of barnase melting in its complex with barstar is 20 degrees C higher than that of the free enzyme. The barstar melting temperature is almost the same in the complex or alone (71 degrees C at pH 6.2 and 68 degrees C at pH 8.0). It seems possible that when barstar unfolds it can remain bound to barnase, while the latter unfolds only on dissociation of the denatured barstar.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases.

The aim of this work is to determine the conformation of the nucleobase adjacent to the cleavable phosphodiester bond in the productive enzyme-substrate complex of RNA-depolymerizing enzymes. To this end the kinetic parameters of hydrolysis of UpA, 2'-C-Me- and 3'-C-Me-UpA were determined for RNase A, RNase Pb2, nuclease S1 and snake venom phosphodiesterase. In these derivatives the ranges of the allowed orientation of uridine residues are restricted due to the substitution of methyl groups for the ribose hydrogen atoms. The results described demonstrate that the proposed method is of general value for the estimation of the nucleotide glycoside angles in the productive enzyme-substrate complexes.

Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase).

To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km. This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction. The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.

A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.

The rates of enzymatic hydrolysis of 2',5'-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3'-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2'-5')adenylyl(2'-5')adenosine.

Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase.

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40,82Ala mutant are about 10(-12) M, only 5 to 43 times higher than those of the barnase-barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase-barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

Mutational analysis of the active site of RNase of Bacillus intermedius (BINASE).

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101 Glu is 2.0-2.7% of that for the native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant kcat, with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both kcat and KM for poly(A) and poly(A) but in kcat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.

State-of-the-art medical pH probes.

This article deals with the development of reusable peroral, transnasal, and endoscopic pH probes, and it looks into the operational features of pH probes equipped with silver-chloride distal and surface reference electrodes. The results obtained revealed that pH probes with a surface reference electrode offer a number of advantages over pH probes with a distal reference electrode.

Single-strand-preferring RNases degrade double-stranded RNAs by destabilizing its secondary structure.

To establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residues, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U).poly(A), poly(C) and poly(C).poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I).poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U).poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U).poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U).poly(A) are discussed on the basis of their efficient estabilishing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly (U).poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U).poly(A) at physiological ionic strength.

[Interrelation between hemodynamic parameters and the functional state of components of the endocrine system in patients with pheochromocytoma]. / Vzaimosviaz' pokazatelei gemodinamiki i funktsional'nogo sostoianiia nekotorykh zven'ev éndokrinnoi sistemy u bol'nykh feokhromotsitomoi.

Ten patients with pheochromocytoma were examined before operation, 8 were examined 2-4 weeks after surgical treatment, and 30 in the long-term periods after elimination of hypercatecholaminemia. The patients with pheochromocytoma manifested activation of the renin-aldosterone system and inhibition of PGE2 secretion. In the long-term postoperative period, the level of aldosterone in the blood plasma was increased and that of PGE2 was reduced. In the patients with pheochromocytoma, the interrelation was discovered between the magnitude of arterial blood pressure and secretion of catecholamines, deoxycorticosterone, depressor prostaglandins. At different times after surgical treatment the central hemodynamics was found to be related to the level of mineralocorticoid hormones.

A simulation-based approach to forecasting the next great San Francisco earthquake.

In 1906 the great San Francisco earthquake and fire destroyed much of the city. As we approach the 100-year anniversary of that event, a critical concern is the hazard posed by another such earthquake. In this article, we examine the assumptions presently used to compute the probability of occurrence of these earthquakes. We also present the results of a numerical simulation of interacting faults on the San Andreas system. Called Virtual California, this simulation can be used to compute the times, locations, and magnitudes of simulated earthquakes on the San Andreas fault in the vicinity of San Francisco. Of particular importance are results for the statistical distribution of recurrence times between great earthquakes, results that are difficult or impossible to obtain from a purely field-based approach.