Structure of the O-polysaccharide of Hafnia alvei strain PCM 1189 that has hexa- to octasaccharide repeating units owing to incomplete glucosylation

The O-polysaccharide of Hafnia alvei PCM 1189 consists of D-glucose, D-galactose, D-GalNAc and D-GlcA and lacks the strict regularity. The intact and carboxyl-reduced polysaccharides as well as oligosaccharides obtained by partial acid hydrolysis were studied by chemical and enzymatic analyses, methylation and NMR spectroscopy. The following structure was established for the O-polysaccharide, which is built up of branched hexa- to octasaccharide repeating units differing in the number of lateral glucose residues: [structure: see text] where the glucose residues shown in italics are nonstoichiometric substituents. The repeating units include also a minor O-acetyl group, whose position was not determined.     

Activation of human meiosis-specific recombinase Dmc1 by Ca2+

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.     

Thymidine kinase and adenosine kinase activities in homogenates of thyroid lobes in hemithyroidectomized rats; effects of melatonin in vitro

OBJECTIVES: Thymidine kinase (TK, EC 2.7.1.21) is a part of the pyrimidine salvage pathway, involved in DNA synthesis. In turn, adenosine kinase (AK, EC 2.7.1.20) functions as a part of the purine metabolic pathway, involved in DNA synthesis. Melatonin (Mel) is an indoleamine which is known to inhibit growth processes in the thyroid gland and also in other endocrine and non-endocrine tissues. The aim of our study was to examine TK and AK activities in homogenates of the rat thyroid lobes remaining after contralateral hemithyroidectomy (hemiTx); additionally, incubations with Mel (10(-6), 10(-9), and 10(-12) M) were performed. METHODS: The experiment was performed on young male Wistar rats (6-week old). The enzyme activities were measured by ascending chromatography and expressed as the amounts of radioactive reaction products of the phosphorylation of dThd (for TK) and of dAdo (for AK). RESULTS: 1. HemiTx increased TK activity in homogenates of the remaining thyroid lobe; 2. Mel increased TK activity in all the groups (intact, sham-operated- and hemiTx-rats), except for the concentrations of 10(-9) and 10(-12) M in the hemiTx-rats, in which the increasing effects of Mel on TK activity reached the borderline statistical significance only; 3. Mel increased the AK activity in intact and in shamTx animals; 4. No statistically significant changes were found in AK activity following Mel in vitro in the incubated remaining thyroid lobes, collected from hemiTx-rats.     

Role of modified nucleosides of yeast tRNAPhe in ribosomal binding

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.     

Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs

A CpG-containing DNA oligonucleotide functionalized with the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group (CpG ODN fma1555) was prepared from phosphoramidites 1a–d using solid-phase techniques. The oligonucleotide behaved as a prodrug by virtue of its conversion to the well-studied immunomodulatory CpG ODN 1555 through thermolytic cleavage of the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group. Such a conversion occurred at 37°C with a half-time of 73 h. The immunostimulatory properties of CpG ODN fma1555 were evaluated in two in vivo assays, one of which consisted of mice challenged in the ear with live Leishmania major metacyclic promastigotes. Local intradermal administration of CpG ODN fma1555 was as effective as that of CpG ODN 1555 in reducing the size of Leishmania lesions over time. In a different infectious model, CpG ODN 1555 prevented the death of Tacaribe-infected mice (43% survival) when administered between day 0 and 3 post infection. Administration of CpG ODN fma1555 three days before infection resulted in improved immunoprotection (60–70% survival). Moreover, co-administration of CpG ODN fma1555 and CpG ODN 1555 in this model increased the window for therapeutic treatment against Tacaribe virus infection, and thus supports the use of thermolytic oligonucleotides as prodrugs in the effective treatment of infectious diseases.     

The regulation of the interaction between F-actin and muscle fructose 1,6-bisphosphatase

Interaction between rabbit muscle fructose 1,6-bisphosphatase (FBPase) and rabbit muscle F-actin results in heterologous complex formation [A. Gizak, D. Rakus, A. Dzugaj, Histol. Histopathol. 18 (2003) 135]. Calculated on the basis of co-sedimentation-binding experiments and ELISA assay-binding constant (Ka) revealed that FBPase binds to F-actin with Ka equal to 7.4 x 10(4) M(-1). The binding is down-regulated by ligands interacting with the FBPase active site (fructose 6-phosphate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate) and with the FBPase allosteric inhibitory site (AMP). The binding and the kinetic data suggests that FBPase may bind F-actin using a bipartite motif which includes the amino acids residues involved in the binding of the substrate as well as of the allosteric inhibitor of the enzyme. The in situ co-localization experiment, in which FBPase was diffused into skinned muscle fibres pre-incubated with phalloidin (polymeric actin-interacting toxin), has shown that FBPase binds predominantly to the region of the Z-line.     

Parallel solid-phase synthesis and characterization of new sulfonamide and carboxamide proline derivatives as potential CNS agents

A solid-phase synthesis of the 64-member library of novel sulfonamide and carboxamide proline derivatives, focused on the 5-HT7 receptor antagonist SB-258741, was described. The final compounds were obtained in good yields and high purity upon cleavage from SynPhase Lanterns, functionalized by a BAL linker. The library representatives were screened for 5-HT7, 5-HT1A and D2 receptors to explore the impact of a tertiary amine moiety, the length of an alkylene spacer and the aryl fragment on the receptor affinity. The preliminary biological results provided data for further investigation aimed at a search for 5-HT7 receptor agents, and permitted the identification of several compounds with significant 5-HT1A receptor affinity.     

6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid mimics active conformation of tyrosine in opioid peptides

6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (6Htc) has been proposed as a rigid mimic of tyrosine conformation in opioid ligand-receptor complex. The significant receptor binding to mu and delta opioid receptors of respective analogues of deltorphin, dermorphin, and endomorphin with D,L-6Htc prove initial prediction.