Structural basis for metal ion coordination and the catalytic mechanism of sphingomyelinases D

Sphingomyelinases D (SMases D) from Loxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-angstrom resolution using the quick cryo-soaking technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (alpha/beta)(8) barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg2+ ion, which is coordinated by Glu(32), Asp(34), Asp(91), and solvent molecules. In the proposed acid base catalytic mechanism, His(12) and His(47) play key roles and are supported by a network of hydrogen bonds between Asp(34), Asp(52), Trp(230), Asp(233), and Asn(252).

Measurement of B-d mixing using opposite-side flavor tagging

We report on a measurement of the B-d(0) mixing frequency and the calibration of an opposite-side flavor tagger in the D0 experiment. Various properties associated with the b quark on the opposite side of the reconstructed B meson are combined using a likelihood-ratio method into a single variable with enhanced tagging power. Its performance is tested with data, using a large sample of reconstructed semileptonic B ->mu(DX)-X-0 and B ->mu(DX)-X-* decays, corresponding to an integrated luminosity of approximately 1 fb(-1). The events are divided into groups depending on the value of the combined tagging variable, and an independent analysis is performed in each group. Combining the results of these analyses, the overall effective tagging power is found to be epsilon D-2=(2.48 +/- 0.21(-0.06)(+0.08))%. The measured B-d(0) mixing frequency Delta m(d)=0.506 +/- 0.020(stat)+/- 0.016(syst) ps(-1) is in good agreement with the world average value.

Use of vitamin D to reduce lameness in broilers reared in harsh environments

In tropical poultry-producing countries, poultry houses usually have little environmental control. This study investigated the effect of dietary vitamin D on the incidence of leg abnormalities of a fast-growing broiler strain reared under harsh conditions. In this study, 300 one-day-old male broilers were distributed in two treatments with three replicates of 50 birds each. One group was fed a placebo and the other group was fed 25-hydroxycholecalciferol (25-OH-D3) in a soluble form. The environmental variables were weekly recorded during until birds were 49 days old. Birds were weekly gait scored, and their walking speed and vertical force while walking were assessed. Post-mortem examination was performed and skeletal lesions were assessed. Control birds presented more leg problems than those that ingested 25-OH-D3. These results suggest that dietary vitamin D is effective in decreasing the severity of lameness by reducing tibial dyschondroplasia and other leg abnormalities.

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Highlights from the I international symposium of thrombosis and anticoagulation in internal medicine, October 23-25, 2008, Sao Paulo, Brazil

The importance of thrombosis and anticoagulation in clinical practice is rooted firmly in several fundamental constructs that can be applied both broadly and globally. Awareness and the appropriate use of anticoagulant therapy remain the keys to prevention and treatment. However, to assure maximal efficacy and safety, the clinician must, according to the available evidence, choose the right drug, at the right dose, for the right patient, under the right indication, and for the right duration of time. The first International Symposium of Thrombosis and Anticoagulation in Internal Medicine was a scientific program developed by clinicians for clinicians. The primary objective of the meeting was to educate, motivate and inspire internists, cardiologists and hematologists by convening national and international visionaries, thought-leaders and dedicated clinician-scientists in Sao Paulo, Brazil. This article is a focused summary of the symposium proceedings. © Springer Science+Business Media, LLC 2009.

A Phase I clinical trial of lodenafil carbonate, a new phosphodiesterase Type 5 (PDE5) inhibitor, in healthy male volunteers

Lodenafil carbonate is a new phosphodiesterase Type 5 (PDE5) inhibitor used in treatment of erectile dysfunction. Objective: The present study was conducted to evaluate the safety, tolerability, and pharmacokinetics of lodenafil carbonate after administering ascending (1 - 100 mg) single oral doses to healthy male volunteers (n = 33). Methods: The study was an open-label, dose-escalation, Phase I clinical trial involving the administration of single oral doses of lodenafil carbonate. Lodenafil carbonate was administered sequentially, escalating in single doses of 1 mg - 100 mg with a washout period of at least 1 week between each dose. The progression to the next dose was allowed after clinical and laboratory exams, Ambulatory Monitoring of Arterial Pressure (AMAP) without relevant clinical modifications and adverse events without clinical relevancy. Blood samples were collected at pre-dose, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 14, 16, 20 and 24 h post-dosing. Plasma samples for measurement of lodenafil carbonate and lodenafil were analyzed by liquid chromatography coupled to tandem mass spectrometry. Results: No serious adverse events were observed, and none of the subjects discontinued the study due to intolerance. The AMAP measurements, clinical and laboratory exams and ECG revealed no significant changes even at higher doses. Lodenafil carbonate was not detected in any samples, indicating that it acts as a prodrug. The mean lodenafil pharmacokinetic parameters for t(max) and t(1/2) were 1.6 (+/- 0.4) h and 3.3 (+/- 1.1) h, respectively. This study demonstrated that lodenafil carbonate was well tolerated and showed a good safety profile in healthy male volunteers.

The impact of R&D in firm value across Europe

In this thesis the impact of R&D expenditures on firm market value and stock returns is examined. This is performed in a sample of European listed firms for the period 2000-2009. I apply different linear and GMM econometric estimations for testing the impact of R&D on market prices and construct country portfolios based on firms’ R&D expenditure to market capitalization ratio for studying the effect of R&D on stock returns. The results confirm that more innovative firms have a better market valuation,investors consider R&D as an asset that produces long-term benefits for corporations. The impact of R&D on firm value differs across countries. It is significantly modulated by the financial and legal environment where firms operate. Other firm and industry characteristics seem to play a determinant role when investors value R&D. First, only larger firms with lower financial leverage that operate in highly innovative sectors decide to disclose their R&D investment. Second, the markets assign a premium to small firms, which operate in hi-tech sectors compared to larger enterprises for low-tech industries. On the other hand, I provide empirical evidence indicating that generally highly R&D-intensive firms may enhance mispricing problems related to firm valuation. As R&D contributes to the estimation of future stock returns, portfolios that comprise high R&D-intensive stocks may earn significant excess returns compared to the less innovative after controlling for size and book-to-market risk. Further, the most innovative firms are generally more risky in terms of stock volatility but not systematically more risky than low-tech firms. Firms that operate in Continental Europe suffer more mispricing compared to Anglo-Saxon peers but the former are less volatile, other things being equal. The sectors where firms operate are determinant even for the impact of R&D on stock returns; this effect is much stronger in hi-tech industries.

I beni immateriali nella valutazione d'azienda

In seguito ad una disamina del materiale presente in letteratura, ci siamo chiesti se i numerosi investimenti pubblicitari, promozionali, di marketing e, per dirla in una parola sola “intangibili”, generassero un aumento del valore dell’impresa nel contesto valutativo oppure se dessero origine esclusivamente ad aumenti di fatturato. L’obiettivo più ambito consiste nel capitalizzare gli investimenti su attività intangibili come la costruzione del marchio, l’utilizzo di brevetti, le operazioni rivolte alla soddisfazione del cliente e tutto quanto si possa definire immateriale. Eppure coesistono nel mare magnum della stessa azienda. Fino a quando non si potrà inserire criteri di valutazione d���azienda delle performance di marketing non ci potrà essere crescita in quanto, le risorse, sono utilizzate senza un criterio di ritorno di investimento.

Au(I) Catalyzed Manipulation of Propargylic Alcohols: A New Route Towards the Synthesis of Indole Alkaloids

In this work we presented several aspects regarding the possibility to use readily available propargylic alcohols as acyclic precursors to develop new stereoselective [Au(I)]-catalyzed cascade reactions for the synthesis of highly complex indole architectures. The use of indole-based propargylic alcohols of type 1 in a stereoselective [Au(I)]-catalyzed hydroindolynation/immiun trapping reactive sequence opened access to a new class of tetracyclic indolines, dihydropyranylindolines A and furoindolines B. An enantioselective protocol was futher explored in order to synthesize this molecules with high yields and ee. The suitability of propargylic alcohols in [Au(I)]-catalyzed cascade reactions was deeply investigated by developing cascade reactions in which was possible not only to synthesize the indole core but also to achieve a second functionalization. Aniline based propargylic alcohols 2 were found to be modular acyclic precursors for the synthesis of [1,2-a] azepinoindoles C. In describing this reactivity we additionally reported experimental evidences for an unprecedented NHCAu(I)-vinyl specie which in a chemoselective fashion, led to the annulation step, synthesizing the N1-C2-connected seven membered ring. The chemical flexibility of propargylic alcohols was further explored by changing the nature of the chemical surrounding with different preinstalled N-alkyl moiety in propargylic alcohols of type 3. Particularly, in the case of a primary alcohol, [Au(I)] catalysis was found to be prominent in the synthesis of a new class of [4,3-a]-oxazinoindoles D while the use of an allylic alcohol led to the first example of [Au(I)] catalyzed synthesis and enantioselective functionalization of this class of molecules (D*). With this work we established propargylic alcohols as excellent acyclic precursor to developed new [Au(I)]-catalyzed cascade reaction and providing new catalytic synthetic tools for the stereoselective synthesis of complex indole/indoline architectures.

Glucagon, catecholamine and pancreatic polypeptide secretion in type I diabetic recipients of pancreas allografts

Successful pancreas transplantation in type I diabetic patients restores normal fasting glucose levels and biphasic insulin responses to glucose. However, virtually no data from pancreas recipients are available relative to other islet hormonal responses or hormonal counterregulation of hypoglycemia. Consequently, glucose, glucagon, catecholamine, and pancreatic polypeptide responses to insulin-induced hypoglycemia and to stimulation with arginine and secretin were examined in 38 diabetic pancreas recipients, 54 type I diabetic nonrecipients, and 26 nondiabetic normal control subjects. Glucose recovery after insulin-induced hypoglycemia in pancreas recipients was significantly improved. Basal glucagon levels were significantly higher in recipients compared with nonrecipients and normal subjects. Glucagon responses to insulin-induced hypoglycemia were significantly greater in the pancreas recipients compared with nonrecipients and similar to that observed in control subjects. Glucagon responses to intravenous arginine were significantly greater in pancreas recipients than that observed in both the nonrecipients and normal subjects. No differences were observed in epinephrine responses during insulin-induced hypoglycemia. No differences in pancreatic polypeptide responses to hypoglycemia were observed when comparing the recipient and nonrecipient groups, both of which were less than that observed in the control subjects. Our data demonstrate significant improvement in glucose recovery after hypoglycemia which was associated with improved glucagon secretion in type I diabetic recipients of pancreas transplantation.

Effect of oral D-tagatose on liver volume and hepatic glycogen accumulation in healthy male volunteers

Standard toxicity tests with high levels of D-tagatose showed a reversible enlargement of the liver in Sprague-Dawley rats without increase of liver enzymes. The present study tests the hypotheses that partial substitution of dietary sucrose by D-tagatose for 28 days increases the volume of human liver and the concentration of liver glycogen. Twelve healthy, male volunteers were studied in a double-blind crossover study with ingestion of D-tagatose (3x15 g daily) and placebo (sucrose, 3x15 g daily) for periods of 28 days each. Liver volume and glycogen concentration have been determined by magnetic resonance (MR) imaging and spectroscopy, which were accompanied by routine medical examinations. MR examinations before and after the treatments revealed no effects (P>0.05) of treatment, period, or subject for changes in liver volume or glycogen concentration. A steady increase of liver volumes, independent of the D-tagatose or placebo intake, has been observed over the study in parallel with a slight increase in body weight. The treatment with D-tagatose was not associated with clinically relevant changes of the examined clinico-chemical and hematological parameters, including liver enzymes and uric acid.

Insulin-like growth factor I improves aspects of mycophenolate mofetil-impaired anastomotic healing in an experimental model

BACKGROUND: Patients taking immunosuppressants after transplantation may require intestinal surgery. Mycophenolate mofetil (MMF) has been found to impair the healing of colonic anastomoses in rats. This study examined whether insulin-like growth factor (IGF) I prevents MMF impairment of anastomotic healing. METHODS: Sixty-three rats were divided into three groups (MMF, MMF/IGF and control). Animals underwent a sigmoid colon anastomosis with a 6/0 suture, and were killed on days 2, 4 and 6 after surgery. Investigations included bursting pressure measurement, morphometric analysis, and assessment of mucosal proliferation by 5-bromo-2'-deoxyuridine and Ki67 immunohistochemistry of the anastomoses. RESULTS: The leak rate was three of 21, one of 20 and two of 20 in the MMF, MMF/IGF-I and control groups respectively. Anastomotic bursting pressures were significantly lower in the MMF group than in the control group on days 2 and 4, but there was no significant difference by day 6. Values in the MMF/IGF-I and control groups were similar. Colonic crypt depth was significantly reduced in MMF-treated animals on days 2 and 4, but this impairment was attenuated by IGF-I on day 4. Similarly, IGF-I reduced the negative impact of MMF on mucosal proliferation on days 2 and 6. CONCLUSION: Exogenous IGF-I improves some aspects of MMF-impaired anastomotic healing.

Biostratigraphical investigations of Lago d'Averno, near Naples

Remains of diatoms, molluscs, ostracods, foraminifera and pollen exines preserved in the sediments of Lago d'Averno, a volcanic lake in the Phlegrean Fields west of Naples, allowed us to reconstruct the changes in the ecological conditions of the lake and of the vegetation around it for the period from 800 BC to 800 AD. Lago d'Averno was at first a freshwater lake, temporarily influenced by volcanic springs. Salinity increased slowly during Greek times as a result of subsidence of the surrounding land. Saline conditions developed only after the lake was connected with the sea by a canal, when Portus Julius was built in 37 BC. The first post-Roman period of uplift ended with a short freshwater phase during the 7th century after Christ. Deciduous oakwoods around the lake was transformed into a forest of evergreen oaks in Greek times and thrived there - apparently almost uninfluenced by man - until it was felled, when the Avernus was incorporated into the new Roman harbour in 37 BC, to construct a shipyard and other military buildings there. Land-use was never more intense than during Roman times and weakest in Greek and Early Roman times, when the Avernus was considered a holy place, the entrance to the underworld.