Saturated C21–C33Hydrocarbons Are Involved in the Self-Regulation of Pseudomonas fluorescensAdhesion to a Glass Surface

One of the two putative groups of antiadhesins was identified in Pseudomonas fluorescensby the method of gas chromatography–mass spectrometry. A mixture of high-molecular unbranched hydrocarbons (HC) with a chain length from 21 to 33 carbon atoms reduced cell adhesion to a glass surface. These HC accumulated in the culture liquid to a total concentration of 10–15 g/l; the concentrations of individual HC ranged from 0.1 to 3.0 g/l. After the addition of individual HC to the bacterial culture, the number of cells attached to the glass surface decreased. This decrease in cell adhesion was due to the enhanced aggregation of the bacterial cells, which promoted mechanical (hydrodynamic) cell detachment from the surface.     

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.     

Changes in phytoperiphyton community during seasonal succession: influence of plankton sedimentation and grazing by phytophages--Chironomid larvae

The investigation of seasonal changes in spatial structure of phytoperiphyton during succession was conducted at the lower reaches of Akulovsky water channel from April to August 2000. At the beginning of succession from April to June dominant forms were chain-forming diatoms and filamentous green algae, sedimented from plankton. Later, at the middle of June under increasing pressure of herbivorous, they were replaced by stretched unicellular diatoms and colonial cyanobacteria. In late June-August, when herbivorous predation was the most intensive, the relative abundance of typical periphytonic forms decreased while that of settled planktonic forms increased. The effect of planktonic algae sedimentation on periphyton composition was evaluated as similarity between phytoperiphyton and phytoplankton communities measured with Chekanovski--Sorensen index. The value of this index tends to decrease with the development of periphyton while showing some relation to intensity of herbivorous pressure. Minimal values of Chekanovski--Sorensen index were under moderate herbivorous density, whereas maximal values were observed in periods of extremely high or low herbivorous density.     

Electromechanical heterogeneity of the myocardium

Herein we discuss modem data showing that ventricle's working myocardium is highly heterogeneous. Significant transmural differences in electrophysiological and biomechanical properties of cardiomyocytes are reviewed. The reviewed evidence of myocardial heterogeneity constitutes the basis for modem assessment of segmental kinetics of different regions in intact heart. We used muscle duplexes as condensed models of a heterogeneous myocardial system. Experimental data, presented here were obtained both in biological duplexes formed by isolated myocardial preparations and in mathematical models of muscle duplexes. We showed that specific functional heterogeneity of cardiomyocytes, related to their excitation sequence, allowed the myocardium to optimise its contractile function and smooth dispersion of repolarisation.     

Properties of antigenomic hepatitis delta virus ribozyme cis- and trans- analogs

A series of permuted variants of antigenomic HDV ribozyme and trans-acting variants were constructed. The catalytic activity study of the ribozymes has shown that all the variants were capable of self-cleaving with equally biphasic kinetics. Ribonuclease and Fe(II)-EDTA cleavage have provided evidence that all designed ribozymes fold according to the pseudoknot model and the conformations of the initial and cleaved ribozyme are different. A scheme of HDV ribozyme self-cleavage reaction was suggested. The role of hydrogen bonds in the reaction was evaluated by substitution of ribose in the ribozyme for deoxyribose. It was found that the 2'-OH group of U23 and C27 is critical for the reaction to occur; the 2'-OH group of U32 and U39 is important, while 2'-OH groups of other nucleotides of loop 3, stem 4 and stem 1 are unimportant for the cleavage activity.     

Properties of Antigenomic Hepatitis Delta Virus Ribozyme That Consists of Three RNA Oligomer Strands

A three-strand ribozyme, a derivative of antigenomic hepatitis delta virus (HDV) ribozyme, which consists of subfragments of 16 (L), 17 (S), and 33 nucleotides (B), has been constructed. The ternary B-L-S complex formed by the subfragments in stoichiometric ratio was able to catalyze a self-cleavage reaction. Kinetics of this reaction exhibited biphasic behavior and the same parameters as in the case of natural cis-ribozyme. Study of kinetics of reaction initiated by adding various reaction components and the study of binary complex formation between subfragments B and L, B and S, and also ternary B-L-S complex formation revealed that: 1) in the presence of Mg2+, B and S form a stoichiometric complex, L and S do not form complex at all, while B and L form 2 types of complexes, probably B-L and 2B-L; and addition of S subfragment prevented the formation of the latter complex; 2) the reaction initiated by S subfragment proceeds much slower than that initiated by other components pointing to the possibility that in the absence of S L may form a nonproductive complex with B, which is slowly displaced by S followed by productive ternary complex formation. Dissociation constants for binary B-L, B-S and ternary B-L-S complexes have been estimated.     

MONTE: An automated Monte Carlo based approach to nuclear magnetic resonance assignment of proteins

A general-purpose Monte Carlo assignment program has been developed to aid in the assignment of NMR resonances from proteins. By virtue of its flexible data requirements the program is capable of obtaining assignments of both heavily deuterated and fully protonated proteins. A wide variety of source data, such as inter-residue scalar connectivity, inter-residue dipolar (NOE) connectivity, and residue specific information, can be utilized in the assignment process. The program can also use known assignments from one form of a protein to facilitate the assignment of another form of the protein. This attribute is useful for assigning protein-ligand complexes when the assignments of the unliganded protein are known. The program can be also be used as an interactive research tool to assist in the choice of additional experimental data to facilitate completion of assignments. The assignment of a deuterated 45 kDa homodimeric Glutathione-S-transferase illustrates the principal features of the program.     

Turbidimetric study of latex agglutination in 2,4-dichlorophenoxyacetic acid analysis

By the example of 2,4-dichlorophenoxyacetic acid (2,4-D) assay by the inhibition of latex agglutination, new synthetic polymer microspheres for the conjugation with antibodies to 2,4-D and agglutinators based on ovalbumin and polyacrylamide were developed and characterized. The effect of various parameters (the concentration of reagents, the type and the degree of modification of the microsphere surface, and the nature of the carrier in the composition of the agglutinator) on the rate of agglutination and the maximal optical absorption observed during the reaction were studied by turbidimetry. The optimal parameters were found for the assay of 2,4-D by the inhibition of latex agglutination with turbidimetric registration of the results.     

The conserved CPH domains of Cul7 and PARC are protein-protein interaction modules that bind the tetramerization domain of p53

Cul7 is a member of the Cullin Ring Ligase (CRL) family and is required for normal mouse development and cellular proliferation. Recently, a region of Cul7 that is highly conserved in the p53-associated, Parkin-like cytoplasmic protein PARC, was shown to bind p53 directly. Here we identify the CPH domains (conserved domain within Cul7, PARC, and HERC2 proteins) of both Cul7 and PARC as p53 interaction domains using size exclusion chromatography and NMR spectroscopy. We present the first structure of the evolutionarily conserved CPH domain and provide novel insight into the Cul7-p53 interaction. The NMR structure of the Cul7-CPH domain reveals a fold similar to peptide interaction modules such as the SH3, Tudor, and KOW domains. The p53 interaction surface of both Cul7 and PARC CPH domains was mapped to a conserved surface distinct from the analogous peptide-binding regions of SH3, KOW, and Tudor domains, suggesting a novel mode of interaction. The CPH domain interaction surface of p53 resides in the tetramerization domain and is formed by residues contributed by at least two subunits.     

The preparation of an IgG diagnostic agent based on stained polyacrolein latexes for use in the latex agglutination reaction

IgG diagnosticum for measuring the concentration of 131I-labeled IgG antibodies to enteric antigen beta 1MA by the latex agglutination inhibition (LAI) test has been prepared on the basis of polyacrolein latexes. A method for the titration of anti-IgG antibodies with the use of the above diagnosticum has been developed, based on the late, agglutination (LA) test. The optimum conditions for the microtitration variant of the LA and LAI tests have been defined. High sensitivity, specificity and simplicity of analysis with the use of latex IgG diagnosticum have been demonstrated. The newly developed methods have been successfully used in laboratory trials of a new diagnostic radiopharmaceuticals for the assay of 131I-labeled antibodies in this preparation and for the detection of side effects of immunization on the recipients.