Sodium periodate/TEMPO as a selective and efficient system for amine oxidation.

A new metal-free protocol for promoting oxidation of amines in aqueous-organic medium was developed. NaIO4 and TEMPO as the catalyst emerged as the most efficient and selective system for oxidation of differently substituted benzyl amines to the corresponding benzaldehydes without overoxidation. Unsymmetrical secondary amines underwent selective oxidation only at the benzylic position thus realising an oxidative deprotection of a benzylic group with an easy amine recovery.

Impact of the new european paediatric regulatory framework on ethics committees: overview and perspectives.

AIM: To evaluate the impact of the new European paediatric regulatory framework on the activities of Ethics Committees operating in Europe and to assess their involvement and interest in paediatric research. METHODS: Task-force in Europe for Drug Development for the Young Network of Excellence and Relating Expectations and Needs to the Participation and Empowerment of Children in Clinical Trials project set up an inventory of Ethics Committees existing in Europe and conducted a survey on their approach to paediatric trials. RESULTS: Ethics Committees operating in 22 European Countries participated in this survey. Results showed a high lack of knowledge, understanding and awareness of the current European paediatric regulatory framework and a lack of involvement of Ethics Committees in paediatric research, especially in terms of training and education, demonstrated also by the decreasing number of Ethics Committees answering exhaustively to the whole questionnaire. The majority of participating Ethics Committees expressed interest in future initiatives related to paediatric research. CONCLUSIONS: Despite a limited knowledge and understanding of the current paediatric regulatory framework, a significant number of Ethics Committees operating in Europe show interest in initiatives related to paediatric research. Networking may be an essential tool to be used to enhance Ethics Committees role in supporting paediatric research. Any initiative should be undertaken at European level in collaboration with European Union Institutions.

Monocyclic ß-lactams: new structures for new biological activities.

The azetidinone core-structure offers a unique approach to the design and synthesis of new derivatives with unique biological properties. During the last two decades researches convincingly demonstrated that the prospect of structural modifications of monocyclic ß-lactams with specific substituents is an effective procedure for the detection and improvement of important pharmacological effects different from antibacterial activity. As a matter of fact, new ß-lactam compounds demonstrated biological activity as inhibitors of a wide range of enzymes. This review reports the latest developments on monocyclic ß-lactam compounds activity as anticancer, antitubercular, HFAAH inhibitors, HDAC inhibitors, anti-inflammatory drugs (tryptase inhibitors), Cathepsin K inhibitors, and vasopressin inhibitors. We attempted to highlight the intertwined relationships between structural features and biological activities, by analysing groups anchored on the three positions of the azetidinone ring as sources of molecular diversity.

BDNF Val66Met polymorphism and brain volumes in multiple sclerosis.

Brain derived neurotrophic factor (BDNF) regulates several CNS physiological and pathological processes. To investigate in multiple sclerosis (MS) patients, the relationship between the Val66Met polymorphism of BDNF and clinical markers of disease activity and MRI markers of focal and diffuse brain pathologies. 45 MS patients and 34 healthy controls (HCs) were genotyped and subjected to clinical-MRI examination. Global white matter fraction (gWM-f), gray matter-f (GM-f), cerebrospinal fluid-f (CSF-f), and abnormal WM-f were measured. We studied 26 Val/Val and 19 Val/Met patients and 23 Val/Val and 11 Val/Met HCs. We found that Val/Val patients had lower GM-f and higher CSF-f than Val/Val HCs; such differences were not statistically significant comparing Val/Met patients to HCs. The regression analysis showed that both Val/Met genotype and relapse number were associated with lower CSF-f. Our data suggest that Met allele might be a protective factor against MS as it is associated to a lower brain atrophy.

Dietary polyphenols: focus on resveratrol, a promising agent in the prevention of cardiovascular diseases and control of glucose homeostasis.

Plants continuously produce an extraordinary variety of biologically active low-molecular-mass compounds. Among them, resveratrol (3,5,4'-trihydroxystilbene) is endowed with significant positive activities by protecting against cardiovascular diseases and preventing the development and progression of atherosclerosis. Furthermore, the molecule significantly ameliorates glucose homeostasis in obese mice. These beneficial effects have driven considerable interest towards resveratrol molecular activities, and intensive efforts for the identification of the stilbene targets have been made. The molecule shows a pleiotropic mode of action. Particularly, its cellular targets are crucial for cell proliferation and differentiation, apoptosis, antioxidant defence and mitochondrial energy production. The complexity of resveratrol activities might account for its effectiveness in ameliorating multifactorial processes, including the onset and/or progression of several degenerative diseases such as myocardial infarction, atherosclerosis and type 2 diabetes. This article reports the actions of resveratrol on cardiovascular diseases and the molecular bases of its activity. We also discuss recent data on the effect of resveratrol on glucose homeostasis and obesity. Finally, the relevance of the stilbene use in the development of new pharmacological strategies is evaluated.

Pharmacokinetics and metabolism of hydroxytyrosol, a natural antioxidant from olive oil.

3,4-Dihydroxyphenylethanol (DOPET) is the major o-diphenol detectable in extra virgin olive oil, either in free or esterified form. Despite its relevant biological effects, mainly related to its antioxidant properties, little data have been reported so far on its toxicity and metabolism. The aim of the present work is to evaluate DOPET toxicity and to investigate its molecular pharmacokinetics by using the (14)C-labeled diphenol. When orally administered to rats, the molecule does not show appreciable toxicity up to 2 g/kg b.wt. To identify and quantify its metabolites, [(14)C]DOPET has been synthesized and intravenously injected in rats. The pharmacokinetic analysis indicates a fast and extensive uptake of the molecule by the organs and tissues investigated, with a preferential renal uptake. Moreover, 90% of the administered radioactivity is excreted in urine collected up to 5 h after injection, and about 5% is detectable in feces and gastrointestinal content. The characterization of the labeled metabolites, extracted from the organs and urine, has been performed by high-pressure liquid chromatography analysis. In all the investigated tissues, DOPET is enzymatically converted in four oxidized and/or methylated derivatives. Moreover, a significant fraction of total radioactivity is associated with the sulfo-conjugated forms, which also represent the major urinary excretion products. On the basis of the reported results, an intracellular metabolic pathway of exogenously administered DOPET, implying the involvement of catechol-O-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase, and phenolsulfotransferase, has been proposed.

Metabolic consequences of hyperhomocysteinemia in uremia.

An elevated blood level of homocysteine (Hcy), a sulfur amino acid, is associated with increased cardiovascular risk. Hcy is generated from S-adenosylhomocysteine (AdoHcy), the demethylated product of S-adenosylmethionine (AdoMet) in transmethylation reactions. AdoHcy is a competitive inhibitor of AdoMet-dependent methyltransferases. AdoHcy accumulation is prevented by rapid metabolism of its products. Chronic renal failure (CRF) is almost constantly associated with hyperhomocysteinemia. It has been shown that: (1) AdoHcy concentration is significantly increased and the AdoMet-AdoHcy ratio is reduced in erythrocytes of patients with CRF; (2) erythrocyte membrane protein methyl esterification, catalyzed by the enzyme protein L-isoaspartyl O-methyltransferase (PCMT; EC 2.1.1.77), is reduced in CRF; PCMT catalyzes a repair reaction involved in the conversion of an isopeptide bond (detrimental to protein structure and function) into a normal peptide bond; (3) D-aspartate residues, a side product of protein methylation and repair, are significantly reduced in erythrocyte membrane proteins of patients with CRF; and (4) folate treatment significantly reduces plasma Hcy levels and improves AdoMet-AdoHcy ratios. Stable isotope studies recently confirmed that the rate of methyl transfer reactions is significantly reduced in uremia. Additional evidence, obtained by independent groups, is consistent with this interpretation. We recently found increased isoaspartyl content of circulating plasma protein levels, particularly albumin, which was only partially reduced after folate treatment, in uremia. This kind of molecular damage possibly is caused by protein increased intrinsic instability as a result of interference with the uremic milieu. In conclusion, Hcy is an uremic toxin involved in protein molecular damage through the inhibition of methylation reactions and protein PCMT-mediated repair.

UVA irradiation induces L-isoaspartyl formation in melanoma cell proteins.

It has been reported that UVA effects are partly mediated by production of reactive oxygen species. Moreover, oxidative stress increases protein damage, involving the occurrence of isoaspartyl residues, a product of protein deamidation/isomerization reactions. This work was undertaken in order to study the effects of UVA irradiation, mediated by oxidation, on sensitive protein targets. Melanoma cells exposed to UVA rays have been chosen as a model for monitoring the occurrence of L-isoaspartyl sites. A dramatic increase of these abnormal residues, specifically recognized and methylated by the enzyme L-isoaspartate(D-aspartate) O-methyltransferase (PCMT; EC 2.1.1.77), can be detected after exposure of M14 cells to raising doses of UVA. The effect of UVA on NO and TBARS accumulation, as well as on DNA fragmentation, has also been investigated. NO formation parallels the increase in isoaspartyl formation, while lipid peroxidation occurs only at the highest UVA doses. No DNA fragmentation has been detected under the employed experimental conditions. These results, as a whole, indicate that protein damages are one of the early events on UVA-induced cell injury. The endogenous activity of PCMT remains remarkably stable under UVA treatment, suggesting that this enzyme might play a crucial role in the repair and/or disposal of damaged proteins in UVA-irradiated cells.

Plasma proteins containing damaged L-isoaspartyl residues are increased in uremia: implications for mechanism.

BACKGROUND: Several alterations of protein structure and function have been reported in uremia. Impairment of a transmethylation-dependent protein repair mechanism possibly related to a derangement in homocysteine metabolism is also present in this condition, causing erythrocyte membrane protein damage. Homocysteine may affect proteins via the accumulation of its parent compound S-adenosylhomocysteine (AdoHcy), a powerful in vivo methyltransferase inhibitor. However, since plasma homocysteine is mostly protein bound, a direct influence on protein structures cannot be ruled out. We measured the levels of L-isoaspartyl residues in plasma proteins of uremic patients on hemodialysis. These damaged residues are markers of molecular age, which accumulate when transmethylation-dependent protein repair is inhibited and/or protein instability is increased. METHODS: L-isoaspartyl residues in plasma proteins were quantitated using human recombinant protein carboxyl methyl transferase (PCMT). Plasma concentrations of homocysteine metabolites were also measured under different experimental conditions in hemodialysis patients. RESULTS: The concentration of damaged plasma proteins was increased almost twofold compared to control (controls 147.83 +/- 17.75, uremics 282.80 +/- 26.40 pmol of incorporated methyl groups/mg protein, P < 0.003). The major protein involved comigrated with serum albumin. Although hyperhomocysteinemia caused a redistribution of thiols bound to plasma proteins, this mechanism did not significantly contribute to the increase in isoaspartyl residues. The S-adenosylmethionine (AdoMet)/AdoHcy concentration ratio, an indicator of the flux of methyl group transfer, was altered. This ratio was partially corrected by folate treatment (0.385 +/- 0.046 vs. 0.682 +/- 0.115, P < 0.01), but protein L-isoaspartate content was not. CONCLUSIONS: Plasma protein damage, as determined by protein L-isoaspartyl content, is increased in uremia. This alteration is to be ascribed to an increased protein structural instability, rather than the effect of hyperhomocysteinemia.

Homocysteine and transmethylations in uremia.

Homocysteine is regarded as a cardiovascular risk factor in both the general population and chronic renal failure patients. Among the mechanisms for homocysteine toxicity, its interference with transmethylation reactions, through its precursor/derivative S-adenosylhomocysteine, plays a multifarious role. In uremia, inhibition of S-adenosylmethionine methyl transfer reactions has been reported by independent investigators, using multiple approaches. This has several possible consequences, which can ultimately affect the patient's relative state of health.

Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis.

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.

Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT; EC 2. 1.1.77) catalyses the methyl esterification of the free alpha-carboxyl group of abnormal L-isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions. The study shows that levels of L-isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t-butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane-cytoskeleton network stability. We conclude that the increased formation/exposure of L-isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells.

3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated.

Solvation of the Carbonyl Compound as a Predominant Factor in the Diastereofacial Selectivity of Nucleophilic Addition.

Temperature-dependent selectivity in nucleophilic additions is affected by the solvent. The inversion temperature (marked with arrows in the graph) that appears in the nonlinear Eyring plots of ln (anti/syn) versus temperature for the addition of butyllithium to an O-protected alpha-hydroxy aldehyde 1 does not depend on nucleophiles (nBuLi (black triangle), tBuLi (*)), but on the solvent. Its value can be obtained from a plot of the (13)C NMR chemical shift of C=O versus temperature. TBDMS=tBuMe(2)Si.

Pyrrolidine dithiocarbamate induces apoptosis by a cytochrome c-dependent mechanism.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 microM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. Programmed cell death was demonstrated by biochemical analyses, including the activation of procaspase 3 and the cleavage of poly(ADP-ribose) polymerase (PARP). PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21(CIP1) level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.

Ambamustine in the second-line treatment of patients with small-cell lung cancer: a phase II Fonicap study.

Despite a high probability of response to first-line chemotherapy, most patients with small-cell lung cancer (SCLC) will eventually have progression of their disease because of the development of resistant disease. Second-line testing of new drugs is an accepted research strategy in SCLC. In this context, the Italian Lung Cancer Task Force (FONICAP) has tested a new synthetic bifunctional alkylating agent, Ambamustine, with preliminary evidence of activity in other solid tumors. Patients with measurable SCLC, progressive after one first-line chemotherapy regimen (either "sensitive" or "refractory"), were eligible for the study. Ambamustine was administered at the dose of 2 mg/kg as a 1-hour intravenous infusion on day 1 every 21 days. The dose was to be increased to 3 mg/kg if no grade IV toxicity and complete hematologic recovery had occurred by day 22. Sample size was calculated according to a two-stage optimal Simon's design. Seventeen patients were entered into the study. Twelve patients were refractory to prior chemotherapy; 12 had extensive disease; the median age was 64 years (range: 46-75 years) and the median performance status was 1. Among 13 patients who received more than one cycle, 9 patients could increase Ambamustine dose from 2 to 3 mg/kg. No objective response was observed: one patient obtained a 50% regression of the primary tumor with contemporary disease progression in the liver and was qualified as having progressive disease. The treatment was well tolerated: grade IV leukopenia occurred in only 1 patient; grade III anemia occurred in 17.6%, grade III leukopenia in 11.8%, and grade III thrombocytopenia in 23.5%. Nonhematologic toxicity was minimal. Ambamustine, at the dose and schedule used in this study, is well tolerated in pretreated patients with SCLC but has no significant antitumor activity in this unfavorable group of patients.

[Pharmaco-epidemiological study on the prophylactic treatment of migraine. National inquiry on attitude to prescription practices by primary care physicians and neurologists in Franceø]. / Etude pharmaco-épidémiologique sur le traitement de fond de la migraine.

A pharmacoepidemiological survey was conducted in order to understand the pattern of migraine prophylactic drug utilization by French physicians. Neurologists and primary care physicians completed a phone-mail-phone questionnaire which inquired about migraine prophylactic treatment. French neurologists and PCP made the same use of migraine prophylaxis in terms of indication, time interval between treatment onset and evaluation, and duration. The two most commonly chosen migraine prophylactic agents were dihydroergotamine and beta-blockers. This study also showed the importance of considering quality of life to evaluate efficacy of migraine prophylaxis.

Homocysteine, a new crucial element in the pathogenesis of uremic cardiovascular complications.

Most large observational studies available today establish that moderate hyperhomocysteinemia, either genetically or nutritionally determined, is an independent risk factor for myocardial infarction, stroke, and thromboembolic disease. This is also true for chronic renal failure patients, who exhibit a high prevalence of hyperhomocysteinemia (85-100%), which reaches high plasma concentrations (20-40 microM, while control values range between 8 and 12 microM). After a renal transplant, homocysteine levels decrease, but tend to be higher than normal. The cause of hyperhomocysteinemia in renal failure is still obscure, since recent data have questioned the previous notion that a net homocysteine renal extraction and/or excretion take place in man. No matter the cause of its increase, the sulfur amino acid homocysteine is thought to induce an increment in cardiovascular risk through three basic biochemical mechanisms: (1) homocysteine oxidation, with H2O2 generation; (2) hypomethylation through S-adenosylhomocysteine accumulation, and (3) protein acylation by homocysteine thiolactone. The final result is membrane protein damage, endothelial damage, and endothelial cell growth inhibition, among other effects. Hyperhomocysteinemia, in general, is susceptible of therapeutic intervention with the vitamins involved in its metabolism. Depending on the cause, vitamin B6, vitamin B12, betaine, and/or folic acid can be effectively utilized. Chronic renal failure patients benefit from folic acid in high dosage: 1-2 mg are usually not effective ('relative folate resistance'), while 5-15 mg reduce homocysteine levels to a 'normative' range (<15 microM) in a substantial group of patients. Good results are also obtained in transplant patients, best with a combination of folic and vitamin B6. The results of the interventional trials focusing on the possible reduction in cardiovascular risk after homocysteine-lowering therapy, both in the general population and in end-stage renal disease, are expected soon, as well as the genetic and biochemical studies in suitable models, with the aim to clarify the cause-effect link suggested by the numerous observational and basic science studies.

Homocysteine and chronic renal failure.

Homocysteine, a sulfur amino acid, is an important methionine derivative, which has been implicated in the pathogenesis of atherothrombosis. Although only observational, epidemiological studies are available at present, the evidence of an association between hyperhomocysteinemia and increased cardiovascular risk is quite strong and this is confirmed also in a population of chronic renal failure patients. From a biochemical standpoint at least three mechanisms have been summoned so far in order to explain homocysteine toxicity including: oxidation, hypomethylation, and acylation. Proteins are believed to play a crucial role as homocysteine molecular targets. Interference with the functions of several of such macromolecules has been so far described being mediated by any of the above mechanisms. Vitamins may positively influence homocysteine metabolism, thus facilitating the metabolic clearance of this compound. Therefore they are presently considered as potential means for reducing plasma levels of this amino acid and preventing vascular occlusions in hyperhomocysteinemic patients. These compounds, with special regard to folate, are eligible for interventional clinical trials, from which the definitive answer on the role of homocysteine in atherothrombosis is expected.