Ionization constants and solubility of compounds involved in enzymatic synthesis of aminopenicillins and aminocephalosporins

The article deals with experimental determination of ionization constants and solubility for the compounds (target products, initial β-lactams, acylating agents and by-products) involved in enzymatic synthesis of some therapeutically used aminopenicillins and aminocephalosporins, namely ampicillin, amoxicillin, cephalexin, cephadroxil, cephaloglycin, cefaclor, cefprozil, cefatrizine. Methodology of investigations and the evaluation of experimental data for the determination of ionization constants and solubility of the different type electrolytes are presented. Applications of the methods based on acid–base potentiometric titration and on determination of solubility–pH dependence of assayed substances are discussed. The original data on ionization constants and solubility of amoxicillin, cefprozil, cefatrizine, cephadroxil and initial β-lactams for production of cefaclor, cefprozil and cefatrizine, as well as solubility of by-product d-(−)-p-hydroxyphenylglycine are presented. Experimentally determined parameters and constants available in the literature for all abovementioned aminopenicillins and aminocephalosporins are collected. These data might be used for choice of the conditions of both processes: the enzymatic synthesis and the isolation of the product from reaction mixture.     

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis

Introduction

Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA).

Methods

Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining.

Results

The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers.

Conclusions

The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.     

A new species of the millipede genus <Emphasis Type="Italic">Lamyctes</Emphasis> Meinert, 1868 (Chilopoda,Lithobiomorpha, Henicopidae) from European Russia

Lamyctes inexpectatus sp. n., characterized by a distinctive shape of the female gonopods, is described from European Russia. The new species is compared with other species of the genus Lamyctes from Russia, and a key to these species is given. Types of the new species are deposited at the Zoological Institute, Russian Academy of Sciences, St. Petersburg.     

ESR signals of NADH and NADPH under illumination

ESR method was applied to investigate the formation of NADH and NADPH free radicals. It was shown that under the action of light (340 nm) in water and other solutions of these compounds a reaction occurred resulting in the formation of free radicals having the typical ESR spectrum. The analysis of the temperature dependence showed the light-induced ESR signals to be registered at −30°C to −120°C, the most intensive ones being observed at −50°C. It was concluded that the observed ESR signals belonged to the products of one-electron oxidation of the coenzymes-free radicals NAD^. and NADP^..     

Comparison of the genome for phylogenetically related bacteriophages phiKZ and EL of Pseudomonas aeruginosa: evolutionary aspects and minimal genome size

Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phiKZ and EL, which belong to a group of (phiKZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phiKZ and EL genomes.     

Molecular architecture of bacteriophage T4

In studying bacteriophage T4—one of the basic models of molecular biology for several decades—there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device—one of the most complex multiprotein components—has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1463–1476.Original Russian Text Copyright © 2004 by Mesyanzhinov, Leiman, Kostyuchenko, Kurochkina, Miroshnikov, Sykilinda, Shneider.     

Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.     

Alpha-amino acid ester hydrolases: Properties and applications

The review describes two major groups of α-amino acid ester hydrolases (AEHs)—enzymes with a similar active center structure, which determines their unique specificity to esters containing an amino group in the α position to the carbonyl. The first group comprises microbial AEHs of the β-lactam acylase type. Technical biocatalysts based on this group of enzymes are used for the production of semi-synthetic amino-β-lactam antibiotics. The second AEH group includes eukaryotic valacyclovirases, which activate in vivo a number of antiviral and anticancer prodrugs. The directed activity of these enzymes is used for the development of target pharmaceutical preparations for the therapy of viral and oncological diseases. The review summarizes and compares the available data on the structure and properties, substrate specificity, and the kinetic parameters of enzymes of these two groups. Experiments identifying the AEH active site and providing the molecular basis for the unique specificity of these enzymes are discussed. The data from the available scientific and patent publications concerning the aminopenicillin and aminocephalosporin synthesis catalyzed by β-lactam acylase AEHs are reviewed and systematized.     

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA

Background  

The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees.     

Kinetic and Thermodynamic Approach to Design of Processes for Enzymatic Synthesis of Betalactams

An optimal way to design an enzymatic process for the production of betalactam antibiotics based on thermodynamic and kinetic studies is described. The study was performed on model reactions involving synthesis of cephalosporin-acids (cephalotin, cefazolin, cefoxitin) using immobilised cephalosporin-acid synthetase from Escherichia coli as biocatalyst, and aminocephalosporins (cephalexin) using immobilised cells of Xanthomonas rubrilineans containing the aminocephalosporin synthetase. The possibility of direct synthesis of cephalotin and cefoxitin was shown, the main equilibrium parameters were determined and the operation conditions were evaluated. The maximum key amino acid conversion to product of approximately 90% for cefoxitin and cephalotin was achieved using initial concentrations of the corresponding key amino acids of 0.05 λM and, respectively, 2-fold and 4-fold molar excess of the carboxylic acids. Cefazolin and cephalexin production by enzymatic synthesis with using of corresponding biocatalyst with a mechanism of action involving the acylenzyme intermediate was shown possible. The kinetic parameters of the process were estimated and the relationship between the maximum antibiotic yield and the initial concentrations of the substrate and nucleophile in the kinetically controlled synthesis was determined. The technologies for cefazolin and cephalexin enzymatic synthesis were designed and the cefazolin technology was optimised. Maximum yields of cefazolin and cephalexin of more than 90% were predicted by the kinetic model using 4-6-fold molar excess of the acylating agents and maximum yields of approximately 85% were achieved in experiments.