A 72-h intervention for improvement of the rate of optimal antibiotic therapy in patients with bloodstream infections.

Antimicrobial stewardship programs are implemented to optimize the use of antibiotics and control the spread of antibiotic resistance. Many antimicrobial stewardship interventions have demonstrated significant efficacy in reducing unnecessary prescriptions of antibiotics, the duration of antimicrobial therapy, and mortality. We evaluated the benefits of a combination of rapid diagnostic tests and an active re-evaluation of antibiotic therapy 72 h after the onset of bloodstream infection (BSI). All patients with BSI from November 2015 to November 2016 in a 1100-bed university hospital in Rome, where an Infectious Disease Consultancy Unit (Unità di Consulenza Infettivologica, UDCI) is available, were re-evaluated at the bedside 72 h after starting antimicrobial therapy and compared to two pre-intervention periods: the UDCI was called by the ward physician for patients with BSI and the UDCI was called directly by the microbiologist immediately after a pathogen was isolated from blood cultures. Recommendations for antibiotic de-escalation or discontinuation significantly increased (54%) from the two pre-intervention periods (32% and 27.2%, p < 0.0001). Appropriate escalation also significantly increased (22.5%) from the pre-intervention periods (8.1% and 8.2%, p < 0.0001). The total duration of antibiotic therapy decreased with intervention (from 21.9 days [standard deviation, SD 15.4] in period 1 to 19.3 days [SD 13.3] in period 2 to 17.7 days in period 3 [SD 11.5]; p = 0.002) and the length of stay was significantly shorter (from 29.7 days [SD 29.3] in period 1 to 26.8 days [SD 24.7] in period 2 to 24.2 days in period 3 [SD 20.7]; p = 0.04) than in the two pre-intervention periods. Mortality was similar among the study periods (31 patients died in period 1 (15.7%), 39 (16.7%) in period 2, and 48 (15.3%) in period 3; p = 0.90). Rapid diagnostic tests and 72 h re-evaluation of empirical therapy for BSI significantly correlated with an improved rate of optimal antibiotic therapy and decreased duration of antibiotic therapy and length of stay.

5 '-Amino-4-imidazolecarboxamide riboside induces apoptosis in human neuroblastoma cells via the mitochondrial pathway.

5'-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5'-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.

Renoprotective effect of renin-angiotensin system block: what has happened?

The Renin Angiotensin System block achieved by current available therapy (angiotensin II antagonism), despite undoubted beneficial effects, is associated to a persistent cardiovascular and renal mortality. During this angiotensin II antagonism, Renin Angiotensin System block is incomplete and angiotensin II break through. The Author analyses possible etiopathogenetic factors. Renin inhibition and Angiotensin-Converting Enzyme type 2 modulation are indicated and discussed as opportunities of Renin Angiotensin System block other than Angiotensin-Converting Enzyme inhibitors and Angiotensin Receptor Blockers, that may render the Renin Angiotensin System block more quiescient.

Effects of dithiothreitol, dithioerythritol and chelating agents on 5'-nucleotidase from bull seminal plasma.

5'-Nucleotidase from bull seminal plasma is inhibited by dithiothreitol and dithioerythritol. These reactives proved to dissociate the dimeric glycoprotein 5'-nucleotidase of Mr 160 000 into two subunits of apparent Mr 80 000, indicating that the subunits are held together by interchain disulfide bridges. HPLC determinations of cysteic acid and carboxymethylcysteine protein derivatives resulted in 50 +/- 3 half-cystine plus cysteine residues, while 1.9 +/- 0.4 free cysteine residues were estimated by HPLC analysis. The enzyme is inhibited by EDTA and EGTA, and the inhibition appears to be of the non-competitive type for both the chelating agents. Experiments for the enzyme activity recovery by MgCl2 and CaCl2 additions, after the EDTA and EGTA treatments in the presence of 8 M urea, are reported.

Isolation and kinetic properties of 5'-nucleotidase from guinea-pig skeletal muscle.

5'-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5'-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg2+. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The Ki values for ADP and ATP are 0.036 and 0.28 microM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation.

Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties.

Deoxyribose 5-phosphate aldolase was purified 41 times from Bacillus cereus induced by growth on deoxyribonucleosides. The purification procedure includes ammonium sulphate fractionation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and preparative electrophoresis on 10% polyacrylamide gel. The enzyme is stable above pH 6.5, but is rapidly inactivated by sulfhydryl reagents. Being insensitive to EDTA, it may be considered as a Class I aldolase. Among a number of compounds tested (including some carboxylic acids, free and phosphorylated pentoses, nucleotides and nucleosides), none has been found to affect the enzyme activity. The enzyme appears to be dimeric, with a subunit Mr of 23,600. A Km of 4.4 x 10(-4) M was calculated for dRib 5-P.

Synergistic action of ADP and 2,3-bisphosphoglycerate on the modulation of cytosolic 5'-nucleotidase.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, can also behave as a phosphotransferase, operating a transfer of phosphate from a nucleoside monophosphate donor to a nucleoside acceptor which, besides a natural nucleoside, can be also an analog. The enzyme activity is stimulated by ADP, ATP and 2,3-bisphosphoglycerate (BPG). The concentration of effector required to attain half maximal activation (A0.5) for the bisphosphorylated compound is in the millimolar range, so that BPG seems to act as a physiological activator of 5'-nucleotidase only in erythrocytes. However, the combination of BPG and ADP brings about a significant increase of their respective affinity for the enzyme, lowering their A0.5 values approx. 4-times. The observation that BPG favors the phosphotransferase more than the hydrolase activity of 5'-nucleotidase stands for a key role of this metabolite in the regulation of the processes of activation of purine pro-drugs, in which this enzyme is involved.

Multiple phosphorylation sites of rat liver glycogen synthase.

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.

Recycling of alpha-D-ribose 1-phosphate for nucleoside interconversion.

Mobilization of the ribose moiety and of the amino group of guanosine may be realized in rat liver extract by the concerted action of purine nucleoside phosphorylase and guanase. Ribose 1-phosphate formed from guanosine through the action of purine nucleoside phosphorylase acts as ribose donor in the synthesis of xanthosine catalyzed by the same enzyme. The presence of guanase, which irreversibly converts guanine to xanthine, affects the overall process of guanosine transformation. As a result of this purine pathway, guanosine is converted into xanthosine, thus overcoming the lack of guanosine deaminase in mammals. Furthermore, in rat liver extract the activated ribose moiety stemming from the catabolism of purine nucleosides can be transferred to uracil and, in the presence of ATP, used for the synthesis of pyrimidine nucleotides; therefore, purine nucleosides can act as ribose donors for the salvage of pyrimidine bases.

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

Purine nucleoside phosphorylase from bovine lens: purification and properties.

Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38,750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97,000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15,250 and 14,650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.

Lens aldo-keto reductase of Camelus dromedarius: purification and properties.

Aldo-keto reductase has been purified 13,000-fold from the lens of the camel (Camelus dromedarius) to a specific activity of 85 U/mg protein. The enzyme is a monomeric protein, exhibiting a Mr = 40,000 upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Camel lens aldo-keto reductase shows a broad substrate specificity, which is strictly dependent on NADPH, and is insensitive to inhibition by Sorbinil and valproate. Aldoses with a carbon chain with more than four residues, as well as glucuronate, are not reduced by the enzyme. On the basis of substrate specificity and sensitivity to inhibition, camel lens aldo-keto reductase appears to be distinct from the so far described aldose, aldehyde and carbonyl reductases.

[Functional renal reserve and glomerular hyperfiltration]. / Riserva funzionale renale e iperfiltrazione glomerulare.

The hemodynamic theory of progressive renal disease with glomerular hyperfiltration has been proposed as common final pathway of kidney disease progression. The evaluation of renal functional reserve is an useful approach to point out glomerular hyperfiltration. First the author describes the meaning of renal functional reserve, mechanisms of glomerular hemodynamic regulation and renal reaction to proteic load and finally explains the practical application of renal functional reserve in clinical nephrology.

[Brain natriuretic peptide and heart failure]. / Peptide natriuretico cerebrale e insufficienza cardiaca.

Heart failure is analysed following the recent classification (prognostic and symptomatic: 4 stages) proposed by the American Heart Association (AHA). The study is focused on the first stage (Stage A) of the AHA classification where the patient is asymptomatic, without heart structural abnormalities but at risk to develop heart failure. In Stage A neurohumural mechanisms of heart failure such as natriuretic peptide system are activated. The author analyses the links between heart failure and the natriuretic peptide system and the role played by the plasma level of brain natriuretic peptide as biomarker of heart failure in Stage A.

The purine analog fludarabine acts as a cytosolic 5'-nucleotidase II inhibitor.

For several years the IMP/GMP-preferring cytosolic 5'-nucleotidase II (cN-II) has been considered as a therapeutic target in oncology. Indeed, various reports have indicated associations between cN-II expression level and resistance to anticancer agents in several cancer cell lines and in patients affected with neoplasia, mainly by hematologic malignancies. In this paper we present evidence showing that, among the commonly used cytotoxic nucleoside analogs, fludarabine can act as a cN-II inhibitor. In vitro studies using the wild type recombinant cN-II demonstrated that fludarabine inhibited enzymatic activity in a mixed manner (Ki 0.5 mM and Ki' 9 mM), whereas no inhibition was observed with clofarabine and cladribine. Additional experiments with mutant recombinant proteins and an in silico molecular docking indicated that this inhibition is due to an interaction with a regulatory site of cN-II known to interact with adenylic compounds. Moreover, synergy experiments between fludarabine and 6-mercaptopurine in human follicular lymphoma (RL) and human acute promyelocytic leukemia (HL-60) cells transfected with control or cN-II-targeting shRNA-encoding plasmids, showed synergy in control cells and antagonism in cells with decreased cN-II expression. This is in line with the hypothesis that fludarabine acts as a cN-II inhibitor and supports the idea of using cN-II inhibitors in association with other drugs to increase their therapeutic effect and decrease their resistance.

Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5'-nucleotidase II.

Cytosolic 5'-nucleotidase II (cN-II) has been reported to be involved in cell survival, nucleotide metabolism and in the cellular response to anticancer drugs. With the aim to further evaluate the role of this enzyme in cell biology, we stably modulated its expression the human glioblastoma cell ADF in which the transient inhibition of cN-II has been shown to induce cell death. Stable cell lines were obtained both with inhibition, obtained with plasmids coding cN-II-targeting short hairpin RNA, and stimulation, obtained with plasmids coding Green Fluorescence Protein (GFP)-fused wild type cN-II or a GFP-fused hyperactive mutant (GFP-cN-II-R367Q), of cN-II expression. Silenced cells displayed a decreased proliferation rate while the over expressing cell lines displayed an increased proliferation rate as evidenced by impedance measurement using the xCELLigence device. The expression of nucleotide metabolism relevant genes was only slightly different between cell lines, suggesting a compensatory mechanism in transfected cells. Cells with decreased cN-II expression were resistant to the nucleoside analog fludarabine confirming the involvement of cN-II in the metabolism of this drug. Finally, we observed sensitivity to cisplatin in cN-II silenced cells and resistance to this same drug in cN-II over-expressing cells indicating an involvement of cN-II in the mechanism of action of platinum derivatives, and most probably in DNA repair. In summary, our findings confirm some previous data on the role of cN-II in the sensitivity of cancer cells to cancer drugs, and suggest its involvement in other cellular phenomenon such as cell proliferation.

The phosphotransferase activity of cytosolic 5'-nucleotidase; a purine analog phosphorylating enzyme.

Cytosolic 5'-nucleotidase is involved in the phosphorylation of several purine nucleoside analogs,used as antiviral and chemotherapeutic agents. In order to assess its role in the mechanisms of activation and inactivation of purine prodrugs, it is essential to study the regulation of both hydrolase and phosphotransferase activities of the enzyme. Using a zone capillary electrophoresis apparatus, we were able to separate substrates and products of the reactions catalyzed by cytosolic 5'-nucleotidase. The method overcomes the frequent unavailability of radiolabeled substrates, and allows the influence of possible effectors and/or experimental conditions on both enzyme activities to be evaluated simultaneously. Results showed that the enzyme was able to phosphorylate several nucleosides and nucleoside analogs with the following efficiency: inosine and 2'-deoxyinosine > 2',3'-dideoxyinosine > 6-chloropurineriboside > 6-hydroxylaminepurine riboside> 2,6-diaminopurine riboside > adenosine > cytidine > deoxycoformycin > 2'deoxyadenosine. This is the first report of deoxycoformycin phosphorylation catalyzed by a 5'-nucleotidase purified from eukaryotic cells. The optimum pH for nucleoside monophosphate hydrolysis was 6.5, slightly more acidic than the optimum pH for the transfer of the phosphate, which was 7.2. Finally, the presence of a suitable substrate for the phosphotransferase activity of cytosolic 5'-nucleotidase caused a stimulation of the rate of formation of the nucleoside. The results suggest the requirements for phosphorylation of nucleoside analogs are a purine ring and the presence of an electronegative group in the 6 position. The stimulation of the rate of nucleoside monophosphate disappearance exerted by the phosphate acceptor suggests that the hydrolysis of the phosphoenzyme intermediate is the rate-limiting step of the process.

5'-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells.

5'-Aminoimidazole-4-carboxamide riboside (AICA riboside) has been previously shown to be toxic to two neuronal cell models [Neuroreport 11 (2000) 1827]. In this paper we demonstrate that AICA riboside promotes apoptosis in undifferentiated human neuroblastoma cells (SH-SY5Y), inducing a raise in caspase-3 activity. In order to exert its effect on viability, AICA riboside must enter the cells and be phosphorylated to the ribotide, since both a nucleoside transport inhibitor, and an inhibitor of adenosine kinase produce an enhancement of the viability of AICA riboside-treated cells. Short-term incubations (2 h) with AICA riboside result in five-fold increase in the activity of AMP-dependent protein kinase (AMPK). However, the activity of AMPK is not significantly affected at prolonged incubations (48 h), when the apoptotic effect of AICA riboside is evident. The results demonstrate that when the cell line is induced to differentiate both toward a cholinergic phenotype (with retinoic acid) or a noradrenergic phenotype (with phorbol esters), the toxic effect is significantly reduced, and in the case of the noradrenergic phenotype differentiation, the riboside is completely ineffective in promoting apoptosis. This reduction of effect correlates with an overexpression of Bcl-2 during differentiation. AICA riboside, derived from the hydrolysis of the ribotide, an intermediate of purine de novo synthesis, is absent in normal healthy cells; however it may accumulate in those individuals in which an inborn error of purine metabolism causes an increase in the rate of de novo synthesis and/or an overexpression of cytosolic 5'-nucleotidase, that appears to be the enzyme responsible for AICA ribotide hydrolysis. In fact, 5'-nucleotidase activity has been shown to increase in patients affected by Lesch-Nyhan syndrome in which both acceleration of de novo synthesis and accumulation of AICA ribotide has been described, and also in other neurological disorders of unknown etiology. Our results raise the intriguing clue that the neurotoxic effect of AICA riboside on the developing brain might contribute to the neurological manifestations of syndromes related to purine dismetabolisms.

Cytosolic 5'-nucleotidase hyperactivity in erythrocytes of Lesch-Nyhan syndrome patients.

Lesch-Nyhan syndrome is a metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch-Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5'-nucleotidase activity in the erythrocytes of seven patients with Lesch-Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5'-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch-Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.