Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.) heterotrophic tissues

Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K _m and V _max values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min⁻¹ mg⁻¹ protein, respectively. The enzyme’s pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells.     

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the “more reactive” and “less reactive” conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.     

Specific calcium antagonist binding sites in brain

The binding of the calcium antagonist [³H] nitrendipine ([³H] NDP) to brain and heart is described and the brain site is characterized. The binding is saturable, specific and of very high affinity with K_D values of 0.16 nM in brain and 0.21 nM in heart. Our kinetic results are similar to those recently reported by two other groups (1,2), indicating a saturable, high affinity binding site in brain. In brain the binding sites are enriched in crude nuclear and synaptosomal fractions. The highest levels of binding are seen in the hippocampus, caudate and cerebral cortex with much lower levels in the cerebellum and pons. Calcium has a marked stimulatory effect on [³H] NDP binding at 10^?4 M. Addition of 0.5 mM CaCl₂ to EDTA treated membranes nearly doubles the number of binding sites. Of the many drugs and neurotransmitters tested only other calcium antagonists, i.e., verapamil, inhibit binding (IC₅₀ = 250 nM). The inhibition of [³H] NDP binding by verapamil is apparently non-competitive and not complete, suggesting that [³H] NDP binds to several sites, only some of which are inhibited by verapamil. The [³H] NDP binding site is probably a protein since it is very sensitive to trypsin, heat and sulfhydryl reagents.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.     

Pyridoxal 5'-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B₆ requiring enzymes. PLP reacts with apo-B₆ enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B₆ enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B₆ proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B₆ enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.     

Amiodarone: a common source of iodine-induced thyrotoxicosis

Amiodarone, a iodine-rich drug widely used in the treatment of tachyarrhythmias, represents one of the most common sources of iodine-induced thyrotoxicosis. The data concerning 58 patients with amiodarone-iodine-induced thyrotoxicosis (AIIT) were analyzed in the present study. Prevalence of AIIT was higher in males than in females (M/F = 1.23/l). Thyrotoxicosis occurred either during treatment with or at various intervals after withdrawal of amiodarone. AIIT developed not only in patients with underlying thyroid disorders, but also in subjects with apparently normal thyroid gland. Classical symptoms of thyrotoxicosis were often lacking, the main clinical feature being a worsening of cardiac disorders. Biochemical diagnosis of AIIT was established by the finding of elevated serum total and free triiodothyronine levels, since elevated serum total and free thyroxine could be found also in euthyroid amiodarone-treated subjects. Twenty-four-hour thyroid radioiodine uptake was very low or undetectable in AIIT patients with apparently normal thyroid glands, while it was inappropriately elevated in patients with underlying thyroid disorders, despite iodine contamination. The role of autoimmune phenomena in the pathogenesis of AIIT appeared to be limited, because circulating thyroid autoantibodies were undetectable in AIIT patients without underlying thyroid disorders or with nodular goiter. Conversely, humoral features of thyroid autoimmunity were mostly found in AIIT patients with diffuse goiter. Treatment of AIIT appeared to be a difficult challenge. Among the 11 patients given no treatment, thyrotoxicosis spontaneously subsided in the 5 patients with apparently normal thyroid gland, whereas the 6 patients with nodular or diffuse goiter were still hyperthyroid 6-9 months after discontinuation of the drug. The administration of high doses (40 mg/day) of methimazole alone proved to be ineffective in most (14/16) patients given this treatment. Twenty-seven patients were treated by methimazole combined with potassium perchlorate (1 g/day). With one exception, euthyroidism was restored within 15-90 days in all cases with underlying thyroid abnormalities, and within 6-55 days in subjects with apparently normal thyroid gland. Thus, the combined treatment appears to be the most effective one, but, due to the potential toxicity of potassium perchlorate, it should be reserved to patients with severe thyrotoxicosis and should be carefully monitored.     

Direct HPLC enantioseparation of chiral aptazepine derivatives on coated and immobilized polysaccharide-based chiral stationary phases

The direct HPLC enantioseparation of Mianserin and a series of aptazepine derivatives is accomplished on polysaccharide-based chiral stationary phases (CSPs). The resolutions are performed on the coated-type Chiralcel OD and Chiralpak AD CSPs and on the first commercially available immobilized-type Chiralpak IA CSP, in normal-phase and polar-organic modes. The complete separation of enantiomers of all racemates investigated was successfully achieved under at least one of CSP/eluent combinations employed. Pure alcohols such ethanol or 2-propanol, with a fixed percentage of DEA added, serve as valuable alternatives to the more common n-hexane-based normal-phase eluents in resolution of Mianserin on the AD CSP. In order to study the chiroptical properties of aptazepine derivatives, chromatographic resolutions are carried out at semipreparative scale using Chiralpak AD and Chiralpak IA as CSPs. Nonconventional dichloromethane-based eluents have permitted to expand the chiral resolving ability of the immobilized Chiralpak IA CSP and to perform mg-scale enantioseparations with an analytical-size column. Assignment of the absolute configuration of the separated enantiomers is empirically established by comparing their chiroptical data with those of structurally related Mianserin.     

Interactions of the Pseudomonas aeruginosa outer membrane proteins with plasma fibronectins. Bacterial adhesin investigation

Pseudomonas aeruginosa adherence is a complex phenomenon largely mediated by pili involving specific receptor-ligand interactions. Anti-fibronectin antibodies as well as plasmatic fibronectin are able to inhibit P. aeruginosa adherence onto A549 cells showing that matricial fibronectin is an actual receptor for this bacterium. Experiments performed in vitro with human plasmatic fibronectin used as receptor and outer membrane proteins of P. aeruginosa as ligands show the presence of four fibronectin-binding proteins. These proteins with molecular mass of 70 +/- 2, 60 +/- 2, 48 +/- 2 and 36 +/- 1 kDa should be adhesins of P. aeruginosa on epithelial cell matrix in a non-pilus mediated adherence.     

Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and l-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.     

Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.