[Causes and predictive factors of prolonged length of hospital stay after flexible ureteroscopy: Experience of a large volume institution]. / Causes et facteurs prédictifs d'une hospitalisation postopératoire prolongée après urétéroscopie souple : expérience d'un centre hospitalo-universitaire.

PURPOSE: To identify the causes of prolonged length of hospital stay (LOHS) of patients treated with flexible ureteroscopy (fURS). The secondary endpoint was to identify the predictors of complications. METHODS: A retrospective single-center cohort study was conducted between January 2011 and December 2015. All consecutive patients treated with a planned fURS, regardless of the indication, AND stayed hospitalized for at least one additional day compared to the traditional postoperative LOHS of our center (=1 day) were included. A multivariate analysis was performed to investigate predictive factors of prolonged LOHS. RESULTS: Two hundred and seventy-two patients were included in the final analysis. The median duration of LOHS was two days [2-18]. Forty-seven percent of patients were discharged beyond day 1 and had no complication. Among them, 56% stayed for simple surveillance decided by the surgeon without any specific treatment introduced and 52% stayed for continuation of intravenous preoperative antibiotics. Of the remaining 144 patients who had a complication, 85.4% (123/144) had a minor complication (Clavien 1 or 2). In multivariate analysis, predictive factors of prolonged LOHS (discharge after day 1) were neurological comorbidities (paraplegia, spina bifida, multiple sclerosis) with an odds ratio of 4.39 [1.7; 11.4]. CONCLUSIONS: The causes of prolonged LOHS were mainly related to comorbidities. A number of patients stayed hospitalized without complications. The identification of predictive factors of complications and prolonged LOHS may allow better selection of patients eligible for outpatient surgery and select those for whom inpatient surgery is still recommended. LEVEL OF EVIDENCE: 3.

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei.

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme-substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈6.1kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240-325cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2-2.3kcal/mol to set the enzyme into a catalytically competent form.

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase.

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer-tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔCp, with NADP binding ΔCp closer to what is expected of crystallographic structures, while NADPH ΔCp is three times larger. The estimated dimer-tetramer association constant is 1.5·10(6)M(-1), and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.

Purinergic modulation of interleukin-1 beta release from microglial cells stimulated with bacterial endotoxin.

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today, 16:524-528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R. A. North and G. Buell, 1996. Science (Wash. DC). 272:735-737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1 beta. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1 beta release triggered by LPS. Our data suggest that LPS-dependent IL-1 beta release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1 beta secretion.

NADPH activates a decarboxylation reaction catalysed by lamb liver 6-phosphogluconate dehydrogenase.

NADP-dependent lamb liver 6-phosphogluconate dehydrogenase catalyses the oxidative decarboxylation of 2-deoxy-6-phosphogluconate, an analogue of the natural substrate. The first products of the reaction are NADPH and 3-keto-2-deoxy-6-phosphogluconate. The NADPH, released from the enzyme, binds to the coenzyme site of the same or the other subunit, activating the decarboxylation reaction in which has not a redox role, since it can be substituted by an analogue devoid of enzymatic redox power. These findings are compared to those obtained with other NADP-dependent decarboxylating dehydrogenases.

Subunits asymmetry in the ternary complex of lamb liver 6-phosphogluconate dehydrogenase detected by a NADP analogue.

Incubation of lamb liver 6-phosphogluconate dehydrogenase, a dimeric enzyme with periodate-oxidized NADP causes the inactivation of the enzyme due to the covalent binding of 2 mol of inhibitor/mol of dimer. In the presence of substrate, the inactivation is faster and is almost complete after the labelling of only one subunit. These results not only confirm the hypothesis of a 'half-of-the-sites' mechanism of action of the enzyme, but also suggest that the formation of the ternary complex (enzyme-substrate-coenzyme) in one subunit causes a conformational change that makes the other subunit unable to bind the coenzyme (and even the adenylic part of it) and, thus, this subunit becomes inactive. It appears that while one subunit catalyses the oxidation of 6-phosphogluconate the other is inactive in this reaction.

6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species.

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

Somatostatin reduces 3H-thymidine incorporation and c-myc, but not thyroglobulin ribonucleic acid levels in human thyroid follicular cells in vitro.

The action of somatostatin (SRIH) on 3H-thymidine (thy) incorporation and on c-myc and thyroglobulin RNA levels in a suspension of follicles from normal and goitrous human thyroid was examined. SRIH, at 10(-7) M concentration, inhibited basal thy incorporation (maximally by 4 h lasting for up 24 h), which effect was greater in goiter than in normal thyroid and was also detected in growing adherent epithelial cells. Moreover, in a follicle suspension SRIH prevented TSH-stimulated thy incorporation, both in normal and in goitrous thyroid. Basal expression of c-myc RNA was not affected by SRIH in either tissue, whereas the TSH-stimulated c-myc RNA level was significantly reduced in goiter. No effect of SRIH was observed on basal or TSH-stimulated thyroglobulin RNA levels. SRIH did not alter basal cAMP concentrations in normal or goitrous follicles, but it significantly reduced TSH-stimulated cAMP accumulation both in normal thyroid and in goiter. Overall, our data indicate a direct inhibitory action of SRIH on growth, but not on differentiation, of human thyroid, probably by a mechanism not entirely cAMP dependent.

In vitro effects of estrogen on tgb and c-myc gene expression in normal and neoplastic human thyroids.

The authors investigated the effects of 17 beta-estradiol (E) and thyroid-stimulating hormone (TSH) on tgb (coding for thyroglobulin), c-myc RNA levels, and [3H]thymidine (thy) incorporation in suspension cultures of normal, adenomatous and carcinomatous human thyroid follicles. The cultured follicles showed decreased tgb RNA and enhanced c-myc RNA levels. In the culture of normal and adenomatous samples E caused a significant increase of [3H]thy incorporation and tgb RNA levels, with no effect on c-myc RNA levels. No effect of E was observed in the carcinomatous thyroid culture. TSH induced a significant increase of [3H]thy incorporation and c-myc expression only in adenoma cultures and a significant increase of tgb RNA levels in both normal and adenomatous samples. TSH had no effect on the carcinoma. The results show that E, like TSH, stimulates in vitro the expression of the tgb gene in differentiated cells, without stimulating the expression of the c-myc proto-oncogene, suggesting a possible action of E on normal thyroid function and perhaps growth, even if not associated with increased c-myc expression.

Glyoxylate for affinity labelling of 6-phosphogluconate dehydrogenase.

In order to find a new reagent for the affinity labelling, 6-phosphogluconate dehydrogenase was treated with glyoxylate, a versatile metabolite with a carboxyl and a reactive aldehydic group. High concentrations of glyoxylate inhibit the enzyme, while in the presence of the reducing agent cyanoborohydride, the enzyme is irreversibly inactivated by only millimolar glyoxylate. This indicates the formation of a Schiff base between the aldehydic group of glyoxylate and one enzyme lysine residue. The kinetics and substrate competition suggest that inactivation is due to affinity labelling. In the first step the inhibitor carboxylic group binds to the substrate carboxyl binding site, and in the second slower step the aldehydic group binds a nearby lysine. We have also found that other enzymes are inactivated by the combined actions of glyoxylate and cyanoborohydride, with a saturation kinetics. Hence, glyoxylate can be helpful to identify specific lysines at the carboxyl binding sites in proteins.

Degradation of aromatic compounds by Trichosporon sp.

When cultured in liquid media, samples of the yeast Trichosporon grow readily and degrade phenol; glutamate was found to stimulate both fungal growth and phenol catabolism, with a distinctive lag. In addition, this same strain grows in the presence of 2-chloro-phenol and 2-methyl, 4-chlorophenol (which are also degraded) and in the presence of 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, which are either degraded or not, as in the case of 4-nitrophenol. The kinetics of both growth and of aromatic catabolism is suggestive of inductive phenomena for key metabolic enzymes.

Identification of the lysine residue involved in the inactivation of lamb liver 6-phosphogluconate dehydrogenase by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate binds almost selectively at the active site of lamb liver NADP-dependent 6-phosphogluconate dehydrogenase causing the inactivation of the enzyme. The substrate and the coenzyme protect against the loss of catalytic activity. The enzyme derivative was digested with trypsin, the labelled peptide was isolated by h.p.l.c. and its amino acid analysis allowed to establish that the inactivator binds to lysine 166 at the active site of the protein.

Use of trinitrobenzensulfonate for affinity labeling of lysine residues at phosphate binding sites of some enzymes.

Trinitrobenzensulfonate, a reagent for lysine residues, inactivates lamb liver 6-phosphogluconate dehydrogenase through affinity labeling. Complete inactivation is due to the binding of only one residue of reagent per enzyme subunit. Other enzymes with a phosphate binding site are also inactivated by affinity labeling. It appears that trinitrobenzensulfonate, when used at low concentrations, first binds to a phosphate binding site, then reacts with a nearby lysine residue. This reagent presents some advantages over pyridoxal phosphate, which has similar characteristics.

Bromopyruvate for the affinity labelling of a single cysteine residue near the carboxylate binding site of lamb liver 6-phosphogluconate dehydrogenase.

Bromopyruvate inactivates 6-phosphogluconate dehydrogenase by affinity labeling. Kinetic analyses, stoichiometry and isolation of a single labelled tryptic peptide of the modified protein indicate that inactivation is due to the affinity labeling of a single cysteine residue, identified as cysteine 401. It thus appears that this cysteine is within a short distance from the protein site involved in the binding of the carboxylate group of the substrate. These results suggest that the carboxylate binding site of proteins could be used as an anchorage point for affinity labeling, and that bromopyruvate can be used to individuate an amino acid residue within few A from this site.

Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor.

The effects of oxidized ATP (oATP) on responses triggered by extracellular adenosine 5'-triphosphate (ATPe) were investigated in the mouse macrophage-like cell line J774. ATPe induced in this cell line two kinds of responses mediated by two different P2 purinergic receptors: 1) an early permeabilization of the plasma membrane to extracellular hydrophilic markers of M(r) up to 900 mediated by P2Z receptors; and 2) a fast mobilization of Ca2+ from intracellular stores mediated by P2Y receptors. Low oATP concentrations (100 microM) completely blocked the first response without affecting the second. ATPe-dependent cell swelling, vacuolization, and lysis were also inhibited. Antagonism developed slowly, as an incubation at 37 degrees C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that the inhibitory action was due to covalent modification of the receptor. The rate of hydrolysis of exogenous ATP was slightly decreased by oATP, indicating a minor blocking effect of this compound on plasma membrane ecto-ATPases in the concentration range tested. These observations suggest that oATP may be a potentially very useful tool for isolation and characterization of the P2Z purinergic receptor.

6-Phosphogluconate dehydrogenase from Trypanosoma brucei. Kinetic analysis and inhibition by trypanocidal drugs.

The kinetics of 6-phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6-phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The T. brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate which have respectively 170-fold and 40-fold higher affinity for the parasite enzyme.

Responses to extracellular ATP of lymphoblastoid cell lines from Duchenne muscular dystrophy patients.

We have observed a striking difference in the response to extracellular ATP in lymphoblastoid cell lines established from Duchenne muscular dystrophy patients and normal subjects. Duchenne muscular dystrophy cells stimulated by extracellular ATP underwent a large increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization, while normal cell lines were little or not at all responsive. These changes in intracellular ion homeostasis were due to activation of an ATP-gated membrane channel permeable to Na+ and Ca2+, with little or no contribution of Ca2+ release from intracellular stores. The channel was selectively activated by ATP, since other purine/pyrimidine nucleotides were ineffective, and it was inhibited by pretreatment with oxidized ATP, a compound previously reported to irreversibly inhibit P2 purinergic receptors. In the presence of extracellular ATP, lymphoblastoid cells established from Duchenne muscular dystrophy patients, but not from healthy controls, underwent rounding and swelling and eventually lysed. The results of this study suggest that lymphoblastoid cells isolated from Duchenne muscular dystrophy patients are eminently sensitive to stimulation by extracellular ATP.

Human leukemia K562 cells: induction to erythroid differentiation by guanine, guanosine and guanine nucleotides.

BACKGROUND AND OBJECTIVE: Human leukemic K562 cells are able to undergo erythroid differentiation in vitro when cultured with a variety of inducers, leading to increased expression of embryo-fetal globin genes such as the zita, epsilon and gamma-globin genes. Therefore the K562 cell line has been proposed as a very useful in vitro model system for determining the therapeutical potential of new differentiating compounds as well as for studying the molecular mechanism(s) that regulate changes in the expression of embryonic and fetal human globin genes. In this study we explored whether nucleoside triphosphates and related compounds are able to induce differentiation of K562 cells. METHODS: K562 cell differentiation was studied using the benzidine test; hemoglobins were characterized by cellulose acetate gel electrophoresis and mRNA accumulation was investigated by Northern blot analysis. RESULTS: The main conclusion of this paper is that guanine, guanosine and guanine ribonucleotides are effective inducers of K562 cell differentiation. Expression of both Hb Portland and Hb Gower 1 is increased in GTP-induced K562 cells. This increase is associated with greater gamma-globin mRNA accumulation. By contrast, ATP, CTP and UTP are not able to induce erythroid differentiation. INTERPRETATION AND CONCLUSIONS: These findings suggest that guanine, guanosine and guanine ribonucleotides are inducers of erythroid differentiation of K562 cells. This is of some relevance since differentiating compounds have been proposed as antitumor agents. In addition, inducers of erythroid differentiation that stimulate gamma-globin synthesis might be considered in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.

The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase.

o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

Active site labeling of erythrocyte transglutaminase by o-phthalaldehyde.

Tissue-type transglutaminase is inactivated in a time-dependent way during incubation with submillimolar concentrations of o-phthalaldehyde, with affinity labeling kinetics. The rate of inactivation by the reagent is greatly enhanced in the presence of the essential enzyme cofactor calcium and is decreased by GTP, an allosteric inhibitor. A fluorescent isoindole derivative is formed during the modification apparently through crosslinkage of active site Cys 277 to a lysine residue. These data and the quenching of fluorescence by addition of calcium ions suggest that the enzyme active site is directly involved in the inactivation process.