The influence of an intramolecular hydrogen bond in differential recognition of inhibitory acceptor analogs by human ABO(H) blood group A and B glycosyltransferases

Human ABO(H) blood group glycosyltransferases GTA and GTB catalyze the final monosaccharide addition in the biosynthesis of the human A and B blood group antigens. GTA and GTB utilize a common acceptor, the H antigen disaccharide alpha-l-Fucp-(1-->2)-beta-d-Galp-OR, but different donors, where GTA transfers GalNAc from UDP-GalNAc and GTB transfers Gal from UDP-Gal. GTA and GTB are two of the most homologous enzymes known to transfer different donors and differ in only 4 amino acid residues, but one in particular (Leu/Met-266) has been shown to dominate the selection between donor sugars. The structures of the A and B glycosyltransferases have been determined to high resolution in complex with two inhibitory acceptor analogs alpha-l-Fucp(1-->2)-beta-d-(3-deoxy)-Galp-OR and alpha-l-Fucp-(1-->2)-beta-d-(3-amino)-Galp-OR, in which the 3-hydroxyl moiety of the Gal ring has been replaced by hydrogen or an amino group, respectively. Remarkably, although the 3-deoxy inhibitor occupies the same conformation and position observed for the native H antigen in GTA and GTB, the 3-amino analog is recognized differently by the two enzymes. The 3-amino substitution introduces a novel intramolecular hydrogen bond between O2' on Fuc and N3' on Gal, which alters the minimum-energy conformation of the inhibitor. In the absence of UDP, the 3-amino analog can be accommodated by either GTA or GTB with the l-Fuc residue partially occupying the vacant UDP binding site. However, in the presence of UDP, the analog is forced to abandon the intramolecular hydrogen bond, and the l-Fuc residue is shifted to a less ordered conformation. Further, the residue Leu/Met-266 that was thought important only in distinguishing between donor substrates is observed to interact differently with the 3-amino acceptor analog in GTA and GTB. These observations explain why the 3-deoxy analog acts as a competitive inhibitor of the glycosyltransferase reaction, whereas the 3-amino analog displays complex modes of inhibition.     

Beta-glucosylation as a part of self-resistance mechanism in methymycin/pikromycin producing strain Streptomyces venezuelae

In our study of the biosynthesis of D-desosamine in Streptomyces venezuelae, we have cloned and sequenced the entire desosamine biosynthetic cluster. The deduced product of one of the genes, desR, in this cluster shows high sequence homology to beta-glucosidases, which catalyze the hydrolysis of the glycosidic linkages, a function not required for the biosynthesis of desosamine. Disruption of the desR gene led to the accumulation of glucosylated methymycin/neomethymycin products, all of which are biologically inactive. It is thus conceivable that methymycin/neomethymycin may be produced as inert diglycosides, and the DesR protein is responsible for transforming these antibiotics from their dormant to their active forms. This hypothesis is supported by the fact that the translated desR gene has a leader sequence characteristic of secretory proteins, allowing it to be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them. Expression of desR and biochemical characterization of the purified protein confirmed the catalytic function of this enzyme as a beta-glycosidase capable of catalyzing the hydrolysis of glucosylated methymycin/neomethymycin produced by S. venezuelae. These results provide strong evidence substantiating glycosylation/deglycosylation as a likely self-resistance mechanism of S. venezuelae. However, further experiments have suggested that such a glycosylation/deglycosylation is only a secondary self-defense mechanism in S. venezuelae, whereas modification of 23S rRNA, which is the target site for methymycin and its derivatives, by PikR1 and PikR2 is a primary self-resistance mechanism. Considering that postsynthetic glycosylation is an effective means to control the biological activity of macrolide antibiotics, the availability of macrolide glycosidases, which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be a valuable part of an overall strategy for the development of novel antibiotics using the combinatorial biosynthetic approach.     

Computed tomography in the evaluation of the bronchopulmonary system in late periods after childhood partial resection of the lung

The authors used computed tomography (CT) as a basis to analyze the anatomic status of the bronchopulmonary system in 28 patients undergone resection of a part of the lung in childhood for chronic nonspecific lung inflammations and malformations. The findings confirmed the high diagnostic value of CT in the evaluation of the lung in different postoperative periods and showed a certain range of changes varying from significant in the lung operated on to progression of the primary disease to the collateral lung.     

Endogenous DNases as a Tool for Isolation of Nuclear Matrix: Critical Parameters of Nucleolysis

The degree of nucleolysis is of critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences, as demonstrated using an example of inactive (c-fos, c-myc, andC ) and active (p53, albumin, and28S rRNA) genes in resting hepatocytes. The optimal degree of nucleolysis is characterized by degradation of loop domains of chromatin, with the relatively uniform molecular weight distribution of DNA being preserved. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched with or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix     

Dependence of immunosuppressive action of lipopolysaccharide on degree of Salmonella pathogenicity

Immunosuppressive activity of Salmonella typhimurium extracellular lipopolysaccharide (LPS) was studied. In this study isogenic S. typhimurium strains with different degree of virulence were used. The attenuation of these strains was linked with mutations on their chromosome (altered synthesis of RNA polymerase or gyrase DNA) or their own virulence plasmid (the insertion of transposon Tn-5). To obtain LPS fraction with different molecular weights, the filtrate of bacterial culture was subjected to gel filtration through a column packed with Sephadex G-200. The immunosuppressive action of LPS fractions was determined on the model of delayed-type hypersensitivity to nonbacterial antigen in experiments on BALB/c mice. The study revealed that transposon-mediated mutation on plasmid, accompanied by the attenuation of salmonellae, led to the loss of immunosuppressive activity of the high-molecular heat-sensitive component of LPS; only the second heat-resistant component with medium molecular weight retained its activity. The presence of two chromosomal attenuating mutations (rifr nalr) was accompanied by the loss of immunosuppressive activity in both components of LPS.     

Genetic structure of Corynebacterium diphtheriae strains isolated in Russia during epidemics of various intensity

The genetic structure of C. dipthteriae toxigenic strains isolated in Russia during the period of more than 50 years was analysed. The use of the method of ribotyping made it possible to register 17 C. diphtheriae ribotypes. The study revealed that the genetic structure of C. diphtheriae population varied in the dynamics of the epidemic process: each epidemic cycle characterized by predominant spread of epidemic strains of definite biovars and ribotypes. Thus, C. diphtheriae strains of biovar gravis, ribotype M11, dominated in the 40-60 years and C. diphtheriae strains of biovar mitis, closely related ribotypes M1 and M1v, dominated in the 80 years. During the last epidemic rise of diphtheriae morbidity in the 90 s C. diphtheriae strains of biovar gravis, closely related ribotypes G1 and G4, dominated among circulating strains. The proportion of these ribotypes began to increase 3 years before the rise of morbidity. The data of microbiological monitoring are recommended for use in the prognostication of the development of the epidemic process of diphtheria infection.     

Structural polymorphism of oligo(dC) with mixed alpha,beta-anomeric backbone

Oligonucleotides with mixed alpha,beta-anomeric backbone have been proposed recently for the recognition of random DNA sequence via new triplex motif (Doronina and Behr, Chem. Soc. Reviews 26, 63-71 (1997)). In the present work we examined alpha- and beta- anomers of cytidine as possible candidates to recognize AT and TA base pairs of the double stranded DNA. The binding properties of beta-oligo(dC) were studied on a series of synthetic oligodeoxynucleotides by UV absorbtion spectroscopy, measurements of bound EtBr fluorescence polarization, circular dichroism (CD) and non-denaturing gel electrophoresis. The UV thermal denaturation, polarization studies and CD experiments with three stranded oligonucleotide 5'-((dCalpha) (dCbeta))5-L-(dAT)5-L-(dAT)5 (L = triethyleneglycol linker) and other oligonucleotide models showed that the formation of semiprotonated oligocytidilic complexes takes place at low temperatures and neutral pH, rather than folding of the clip into intramolecular triplex. The low-temperature transition was observed in denaturation profiles of any oligonucleotide containing beta- or mixed alpha,beta- cytidine stretches at the concentration of 1 microM. Self-association of alpha,beta-oligo(dC) was additionally confirmed by the appearance of two CD bands (at 290 and 265 nm) characteristic of CC+ base pairs. Despite the effective ability of alpha,beta-oligo(dC) to form self-associates, we succeeded in targeting 30-bp AT containing random DNA duplex by a 30-nt alpha,beta-oligocytidilate as evidenced by non-denaturing gel electrophoresis. A complete binding of the duplex was observed at a 5-fold excess of the third strand at 15 degrees C. Along with the formation of the three-stranded complex, self-association of mixed backbone oligo(dC) strands occurred.