Farnesyl pyrophosphate synthase enantiospecificity with a chiral risedronate analog, [6,7-dihydro-5H-cyclopenta[c]pyridin-7-yl(hydroxy)methylene]bis(phosphonic acid) (NE-10501): Synthetic, structural, and modeling studies

The complex formed from crystallization of human farnesyl pyrophosphate synthase (hFPPS) from a solution of racemic [6,7-dihydro-5H-cyclopenta[c]pyridin-7-yl(hydroxy)methylene]bis(phosphonic acid) (NE-10501, 8), a chiral analog of the anti-osteoporotic drug risedronate, contained the R enantiomer in the enzyme active site. This enantiospecificity was assessed by computer modeling of inhibitor–active site interactions using Autodock 3, which was also evaluated for predictive ability in calculations of the known configurations of risedronate, zoledronate, and minodronate complexed in the active site of hFPPS. In comparison with these structures, the 8 complex exhibited certain differences, including the presence of only one Mg²⁺, which could contribute to its 100-fold higher IC₅₀. An improved synthesis of 8 is described, which decreases the number of steps from 12 to 8 and increases the overall yield by 17-fold.     

External respiration function of young male residents of northern Europe during the annual cycle

In young men (19.0 ± 0.9 years of age), the following parameters were studied during the annual cycle: the tidal and minute lung volumes, vital and forced vital capacities of the lungs, expiratory and inspiratory reserve volumes, 0.5-and 1-s forced expiratory volumes, and Tiffenau index. Young men working under the conditions of the North (62°N) proved to have deeper breathing; the minute volume and vital capacity of their lungs were increased. Analysis of the lung volume during the annual cycle demonstrated changes in most parameters studied (except the expiratory reserve volume and Tiffenau index). The maximum values of the lung volumes were recorded in the cold time of the year (from November to April), whereas the minimum values were observed in the warm time (from May to September).     

Single turnover kinetics of phage T4 DNA-(N6-adenine)methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase [EC 2.1.1] was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k1 = 0.21 s-1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k2 = 0.023 s-1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI Mtase. Substitutions of bases and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme-substrate complexes is usually sharply reduced. The flipping of the adenosine residue, a target for the modification in the recognition site, revealed by fluorescence titration, upon interaction with the enzyme supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.     

Structural basis for oligosaccharide recognition by Pyrococcus furiosus maltodextrin-binding protein

A maltodextrin-binding protein from Pyrococcus furiosus (PfuMBP) has been overproduced in Escherichia coli, purified, and crystallized. The crystal structure of the protein bound to an oligosaccharide ligand was determined to 1.85 A resolution. The fold of PfuMBP is very similar to that of the orthologous MBP from E. coli (EcoMBP), despite the moderate level of sequence identity between the two proteins (27 % identity, 46 % similarity). PfuMBP is extremely resistant to heat and chemical denaturation, which may be attributed to a number of factors, such as a tightly packed hydrophobic core, clusters of isoleucine residues, salt-bridges, and the presence of proline residues in key positions. Surprisingly, an attempt to crystallize the complex of PfuMBP with maltose resulted in a structure that contained maltotriose in the ligand-binding site. The structure of the complex suggests that there is a considerable energy gain upon binding of maltotriose in comparison to maltose. Moreover, isothermal titration calorimetry experiments demonstrated that the binding of maltotriose to the protein is exothermic and tight, whereas no thermal effect was observed upon addition of maltose at three temperatures. Therefore, PfuMBP evidently is designed to bind oligosaccharides composed of three or more glucopyranose units.     

Occurrence of Helicobacter pylori specific antigens in diseases of gastrointestinal tract

The results of the immunophoretic analysis of H. pylori antigenic structure are presented. Along with H. pylori O-antigen, specific surface antigen of acidic nature, relatively thermolabile with characteristics similar to those of K-antigens, was detected. A diagnosticum based on the coagglutination test for the rapid detection of H. pylori specific antigens in the patients' biological fluids (coprofiltrate, saliva) by the noninvasive method was developed. The circulation mode of H. pylori in human body and its correlation with the clinical symptoms of the disease was shown. H. pylori antigens were very frequently detected (in 65 - 83% of cases) in patients with acute bacterial enteric infections: shigellosis, salmonellosis, yersiniosis, campylobacteriosis. The newly developed method seems to be promising for the rapid diagnosis of H. pylori associated infections, for the prolonged monitoring and evaluation of the effectiveness of antibacterial therapy.     

Bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase): evidence for two distinct stages of methylation under single turnover conditions

We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA < 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (kmeth1 = 0.21 s-1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (kmeth2 = 0.023 s-1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (kmeth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (kmeth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.     

DNA-[N4-Cytosine]-Methyltransferase from Bacillus amyloliquefaciens: Mechanism of Action Derived from Steady-State Kinetics

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the 5"-GGATCC recognition site catalyzed by the DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens [EC 2.1.1.113] has shown that the dependence of the rate of methylation of the 20-meric substrate duplex on SAM and DNA concentration are normally hyperbolic, and the maximal rate is attained upon enzyme saturation with both substrates. No substrate inhibition is observed even at concentrations many times higher than the K _M values (0.107 M for DNA and 1.45 M for SAM), which means that no nonreactive enzyme–substrate complexes are formed during the reaction. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAMDNAmetDNA SAH (S-adenosyl-L-homocysteine). However, more detailed numerical analysis of the aggregate experimental data admits an alternative order of substrate binding, DNA SAM }, though this route is an order of magnitude slower.     

Comparative study of the M.Bstf5I-1 and M.BstF5I-3 DNA methyltransferases from the Bacillus stearothermophilus F5 restriction-modification system

The BstF5I restriction-modification system from Bacillus stearothermophilus F5, unlike all known restriction-modification systems, contains three genes encoding DNA methyltransferases. In addition to revealing two DNA methylases responsible for modification of adenine in different DNA strands, it has been first shown that one bacterial cell has two DNA methylases, M.BstF5I-1 and M.BstF5I-3, with similar substrate specificity. The boundaries of the gene for DNA methyltransferase M.BstF5I-1 have been verified. The bstF5IM-1 gene was cloned in pJW and expressed in Escherichia coli. Homogeneous samples of M.BstF5I-1 and M.BstF5I-3 were obtained by chromatography with different sorbents. The main kinetic parameters have been determined for M.BstF5I-1 and M.BstF5I-3, both modifying adenine in the recognition site 5'-GGATG-3'.     

Some X-ray diffraction parameters of liquid-crystalline dispersions of nucleic acid-chitosan complexes

Liquid-crystalline dispersions of nucleic acid-chitosan complexes (NA-chitosan) possess optical and X-ray diffraction properties different from those of "classical" cholesterics. It is possible that positive charge distribution (distance between charges, chitosan conformation, etc.) in the polymeric chain of chitosan molecule combining with NA molecule is the factor that affects the spatial structure of resulting dispersions.     

Effects of 460 MHz microwave radiation on Drosophila embryos under raised temperature

Combined effect of 460-MHz microwave irradiation and increased (up to 40 degrees C) temperature on Drosophila embryos of definite age was studied. It was demonstrated that the effect of 5-min exposures to non-modulated microwaves with 6 W/kg SAR accompanied with heating is only a little stronger than at normal temperature (24.5 degrees C). Irradiation with pulse-modulated microwaves with pulse repetition rates of 6, 10, 16, and 22 p.p.s. with average SAR of 0.12 W/kg (pulsed SAR 3 W/kg) combined with increased temperature caused some changes in PID dependent on the pulse rate. At 6 and 22 p.p.s, the increase in PID was close to that observed at normal temperature while at 10 and 16 p.p.s. the microwave irradiation did not produce any noticeable effect on development of the Drosophilas.