Structure of the substrate complex of thymidine kinase from Ureaplasma urealyticum and investigations of possible drug targets for the enzyme

Thymidine kinases have been found in most organisms, from viruses and bacteria to mammals. Ureaplasma urealyticum (parvum), which belongs to the class of cell-wall-lacking Mollicutes, has no de novo synthesis of DNA precursors and therefore has to rely on the salvage pathway. Thus, thymidine kinase (Uu-TK) is the key enzyme in dTTP synthesis. Recently the 3D structure of Uu-TK was determined in a feedback inhibitor complex, demonstrating that a lasso-like loop binds the thymidine moiety of the feedback inhibitor by hydrogen bonding to main-chain atoms. Here the structure with the substrate deoxythymidine is presented. The substrate binds similarly to the deoxythymidine part of the feedback inhibitor, and the lasso-like loop binds the base and deoxyribose moieties as in the complex determined previously. The catalytic base, Glu97, has a different position in the substrate complex from that in the complex with the feedback inhibitor, having moved in closer to the 5'-OH of the substrate to form a hydrogen bond. The phosphorylation of and inhibition by several nucleoside analogues were investigated and are discussed in the light of the substrate binding pocket, in comparison with human TK1. Kinetic differences between Uu-TK and human TK1 were observed that may be explained by structural differences. The tight interaction with the substrate allows minor substitutions at the 3 and 5 positions of the base, only fluorine substitutions at the 2'-Ara position, but larger substitutions at the 3' position of the deoxyribose.     

Transport of malate and citrate into rat brain mitochondria under hypoxia and anesthesia

Hypoxia and anesthesia inhibited penetration of malate and citrate into the brain mitochondria by 60% and 40%, respectively. Anesthetized animals exposed to low oxygen tension showed similar changes as those subjected to hypoxia without anesthesia. Recovery from anesthesia was rapid and the rates of citrate and malate uptake returned to the control values in approximately 60 min. In the case of hypoxia, days were required to restore the control values of citrate and malate uptake. Free fatty acids had no effect on the entry of malate and citrate into the mitochondria. However, changes in the levels of protein—SH groups were observed which may be responsible for the impaired transport of citrate and malate under hypoxic condition.     

Polyamines and Nitric Oxide Link in Regulation of Dormancy Removal and Germination of Apple (Malus domestica Borkh.) Embryos

Polyamines (PAs) belong to plant growth regulators and in complex with classical phytohormones take part in regulation of seed dormancy and germination. Although the impact of reactive oxygen (ROS) and nitrogen (RNS) species on seed germination is well described, the cross talk of PAs with ROS/RNS has never been analyzed. Due to the close connection of PAs and ethylene biosynthetic pathways to arginine (Arg)-dependent NO biosynthesis we investigated production of nitric oxide (NO), peroxynitrite (ONOO^?) and the level of O ₂ ^?? or H₂O₂ in apple embryos, germination of which was PA regulated. PAs: putrescine (Put) and spermidine (Spd) in contrast to spermine (Spm) stimulated germination of apple embryos. Among amino acids, stimulation of germination was observed in Arg and ornithine (Orn) only. Dormancy removal of embryos by PAs was associated with increased accumulation of H₂O₂ and O ₂ ^?? in embryonic axes. At the same stage of completion of sensu stricto germination the stimulatory effect of PAs (Put and Spd) and amino acids, mainly Arg and Orn, was accompanied by enhanced NO and ONOO^? production in embryonic axis. The beneficial effect of PAs (Put and Spd) and their precursors on germination of apple embryos was removed by NO scavenging, suggesting a crucial role of NO in termination of embryo germination and radicle growth. Moreover, activity of polyamine oxidase in embryo axes was greatly enhanced by embryo fumigation with NO. Our data demonstrate the interplay of RNS/ROS with PAs and point to NO action as an integrator of endogenous signals activating germination.     

The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle

Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

Correlation between thyroid stimulating immunoglobulins and thyrotropin binding inhibitory immunoglobulins levels in patients with Graves' disease

INTRODUCTION: The II generation method using human recombination thyrotropin receptors for measurement of thyrotropin binding inhibitory immunoglobulins (TBII) is characterized by increased sensitivity and specificity in comparison with I generation method. AIM OF STUDY was to determine, whether TBII levels measured with II generation assay reflect thyroid stimulation and whether measurement of thyroid stimulating antibodies (TSI) could be replaced by TBII determinations. Specific aim was to evaluate, whether correlation between TSI and TBII levels is stable during antithyroid therapy. MATERIAL AND METHODS: 41 patients with the newly diagnosed Graves' disease were included in the study. TSI (cAMP levels in CHO cell line) and TBII (II generation assay) levels were determined before treatment and after 1, 3, 6, 9 and 12 months of thiamazol therapy. Moreover, thyroid blocking antibodies were determined after 12 months of treatment. RESULTS: 32 patients (82.05%) had positive basic TSI level and 35 patients (89.74%) had positive basic TBII level. After 12 months of therapy negative level of TSI was observed in 67.57% of patients and negative level of TBII was founded in 45.85% of patients. Correlation between TSI and TBII levels was positive during treatment course except time after 9 months of therapy. CONCLUSIONS: TBII level is adequate parameter to assess thyroid stimulation intensity. Positive correlation between TSI and TBII levels is present during almost whole treatment course. TBII seems to be reliable parameter in disease activity monitoring and response to therapy.     

Membrane-bound basic peptides sequester multivalent (PIP2), but not monovalent (PS), acidic lipids

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.     

4-Methylideneisoxazolidin-5-ones--a new class of alpha-methylidene-gamma-lactones with high cytostatic activity

A novel, general method of synthesis of 4-methylideneisoxazolidin-5-ones 10 is described. The target compounds were synthesized starting from ethyl 2-diethoxyphosphoryl-2-alkenoates 6 or dicyclohexylammonium 4-diethoxyphosphoryl-2-alkenoates 7. Addition of N-methylhydroxylamine hydrochloride to these Michael acceptors, lactonization to 4-diethoxyphosphorylisoxazolidin-5-ones 9, and Horner-Wadsworth-Emmons olefination of formaldehyde using 9 gave the title isoxazolidinones 10. All obtained compounds were tested against L-1210, HL-60, and NALM-6 leukemia cell lines. Several isoxazolidinones 10 were found to be very potent with IC(50)<1 microM. The highest cytostatic activity against HL-60 was observed for 10a and against NALM-6 for 10b with IC(50) values of 0.74 and 0.34 microM, respectively.     

Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 ? resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.     

Chitinolytic activity of bacteria

Chitinolytic bacteria play an important role in degradation of chitin, one of the most abundant biopolymers in nature. These microorganisms synthesize specific enzymes, that catalyze hydrolysis of beta-1,4-glycosidic bonds in low-digestible chitin polymers, turning it into low-molecular, easy to digest compounds. During last decades many bacterial chitinolytic enzymes have been studied and characterized, mainly for their potential applications in agriculture, industry and medicine. Several chitinase classifications have been proposed, either on the base of substrate specificity or amino acid sequence similarities. X-ray crystallography and NMR spectroscopy techniques enabled the determination of three dimensional structure of some chitinases, what was helpful in explaining their catalytic mechanism. Development of biotechnology and molecular biology enables a deep research in regulation and cloning of bacterial chitinase genes.