Evidence suggesting interactions between immunodominant membrane protein Imp of Flavescence dorée phytoplasma and protein extracts from distantly related insect species.

AIMS: In this study, binding between the immunodominant membrane protein Imp of the 16SrV-D phytoplasma associated with Flavescence dorée disease (FD-Dp) and insect proteins of vectors and non-vectors of FD-Dp was tested. METHODS AND RESULTS: Six Auchenorrhyncha species, from distantly related groups were selected: Scaphoideus titanus, Euscelidius variegatus, Macrosteles quadripunctulatus, Zyginidia pullula (Cicadomorpha), Ricania speculum and Metcalfa pruinosa (Fulgoromorpha). The vector status of each species was retrieved from the literature or determined by transmission trials in this study. A His-tagged partial Imp protein and a rabbit polyclonal antibody were synthesized and used for Western and Far-Western dot Blot (FWdB) experiments. Total native and membrane proteins (MP) were extracted from entire bodies and organs (gut and salivary glands) of each insect species. FWdB showed decreasing interaction intensities of Imp fusion protein with total proteins from entire bodies of S. titanus, E. variegatus (competent vectors) and M. quadripunctulatus (non-vector), while no interaction signal was detected with the other three species (non-vectors). A strong signal detected upon interaction of FD-D Imp and MP from guts of closely related insects supports the role of this organ as the first barrier to ensure successful transmission. CONCLUSIONS: Our results showed that specific Imp binding, correlated with vector status, is involved in interactions between FD-Dp and insect proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Integrating knowledge on host-pathogen protein-protein interactions and on insect phylogeny would help to identify the actual range of vectors of phytoplasma strains of economic importance.

Combination of gene expression and genome copy number alteration has a prognostic value for breast cancer.

Specific genome copy number alterations, such as deletions and amplifications are an important factor in tumor development and progression, and are also associated with changes in gene expression. By combining analyses of gene expression and genome copy number we identified genes as candidate biomarkers of BC which were validated as prognostic factors of the disease progression. These results suggest that the proposed combined approach may become a valuable method for BC prognosis.

Leg ability factors in tennis players.

We investigated the effects of leg stiffness on running speed, jump height and leg power in 13 male 2(nd)- and 3(rd)-series ranked tennis players (23 ± 3 years old, 73.2 ± 8.4 kg, 1.81 ± 0.06 m). Leg stiffness and jump height were assessed using jumping and hopping tests. Mean running speeds over 20 m and 40 m (speed20 and speed40, respectively) were determined from a sprint test. Theoretical maximal leg power (P(maxth)) was extrapolated from a force-velocity test performed on a cycle ergometer. Leg stiffness averaged 478.7 ± 181.7 N.m⁻¹.kg⁻¹ (34,808 ± 12,573 N.m⁻¹. It was significantly correlated to speed20 and counter movement jump height (r=0.60, P=0.028 and r=0.58, P=0.0407, respectively). There were also significant correlations between P (maxth) and counter-movement jump height (r=0.59, P=0.0335) and between P(maxth) and speed40 (r=0.58, P=0.0393). This study characterizes leg stiffness in tennis players and brings new information concerning the way it is related to several other muscular biomechanical parameters.

Temporal and spatial analysis of astrocyte calcium waves.

In the last decade new ideas were born about the temporal and spatial dynamics of intercellular calcium waves in astrocytes. In this paper we introduce a new approach to analyze the ways in which astrocytes communicate in cultures. We present a method to describe the spatial propagation of Ca(2+) waves in vitro and a technique to compare the activity of different cells in vivo and in vitro under different stimulation conditions. The proposed method resulted to be an interesting way to distinguish different astrocyte clusters, which can be related to the communication characteristics in the network.

Metabolic modulation induced by chronic hypoxia in rats using a comparative proteomic analysis of skeletal muscle tissue.

Hypoxia-induced changes of rat skeletal muscle were investigated by two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The results indicated that proteins involved in the TCA cycle, ATP production, and electron transport are down-regulated, whereas glycolytic enzymes and deaminases involved in ATP and AMP production were up-regulated. Up-regulation of the hypoxia markers hypoxia inducible factor 1 (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) was also observed, suggesting that in vivo adaptation to hypoxia requires an active metabolic switch. The kinase protein, mammalian target of rapamycin (mTOR), which has been implicated in the regulation of protein synthesis in hypoxia, appears unchanged, suggesting that its activity, in this system, is not controlled by oxygen partial pressure.

Protein analysis by capillary zone electrophoresis utilizing a trifunctional diamine for silica coating.

A novel method is here reported for the analysis of mixture of proteins with pI ranging from pH 3-9.5 in an ample pH interval (pH 2.5-9.0) without adsorption onto the naked silica wall. It consists of treating the capillary surface at alkaline pH, typically 9.0, with small amounts (2-4 mM) of a quaternarized piperazine derivative: (N-methyl-N-omega-iodobutyl)-N'-methylpiperazine (Q-PzI). It appears that this compound is able to dock onto the wall via trifunctional links: a salt bridge via the quaternary nitrogen, a hydrogen bond via the tertiary nitrogen, and finally, a covalent link via the terminal iodine in the butyl chain and a neighboring ionized silanol. This last reaction seems to be completed in a few minutes of incubation of the capillary at room temperature. Because the compound is permanently affixed to the wall, its presence is not needed during protein/peptide separations. By properly dosing the level of Q-PzI in the preconditioning step, it is possible to strongly reduce the electroendoosmotic flow (EOF), zero it, or reverse it. Unlike dynamic coatings with oligoamines, which are most effective only at acidic pH values and are required as additives during separations, Q-PzI is effective in an ample pH interval (pH 2.5-9.0) and is not needed during the CZE analysis. A broad pI (pH 3-10) protein mix can be separated according to protein mobility in free phase, suggesting a strong modulating capacity of the functionalized wall. The same separation is not obtained in capillaries permanently coated with neutral, hydrophilic polymers (such as polyacrylamide), even if the quality of a single protein/peptide profile in Q-PzI-conditioned capillaries is equivalent to those obtained in capillaries permanently coated. Although there is strong indirect evidence of the ability of Q-PzI to alkylate the silica wall, to which it is then irreversibly bound, such an alkylation event does not occur with proteins on potentially reacting sites, such as the free -SH of Cys or the -OH group of Tyr, as demonstrated by incubating them overnight in a large molar excess at strongly alkaline pH values and analyzing such proteins by MALDI-TOF mass spectrometry.

Pharmacological and toxicological aspects of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548): a novel antineoplastic agent.

4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548) belongs to a novel class of antitumor compounds (termed alkycyclines) and is currently undergoing Phase II clinical trial. In the present study, we investigated the in vitro and in vivo antitumor activity, the pharmacokinetics, and the toxicological profile of this compound. PNU-159548 showed good cytotoxic activity in murine and human cancer cells growing in vitro, with an average concentration for 50% growth inhibition of 15.8 ng/ml. The drug showed strong antitumor efficacy in vivo after i.v. and p.o. administration against rapidly proliferating murine leukemias and slowly growing transplantable human xenografts. At non-toxic doses, PNU-159548 produced complete regression and cures in ovarian, breast, and human small cell lung carcinomas. Fourteen of 16 models studied, including colon, pancreatic, gastric, and renal carcinomas, astrocytoma and melanoma, were found to be sensitive to PNU-159548. In addition, PNU-159548 was effective against intracranially implanted tumors. Toxicological studies revealed myelosuppression as the main toxicity in both mice and dogs. The maximum tolerated doses, after a single administration, were 2.5 mg/kg of body weight in mice, 1.6 mg/kg in rats, and 0.3 mg/kg in dogs. In the cyclic studies, the maximum tolerated doses were 0.18 mg/kg/day (cumulative dose/cycle: 0.54 mg/kg) in rats and 0.05 mg/kg/day (cumulative dose/cycle: 0.15 mg/kg) in dogs. PNU-159548 showed minimal cardiotoxicity, when compared with doxorubicin in the chronic rat model at a dose level inducing similar myelotoxicity. Animal pharmacokinetics, carried out in mice, rats, and dogs, was characterized by high volumes of distribution, plasma clearance of the same order of the hepatic blood flow, and short terminal half-life. These findings support the conclusion that PNU-159548 is an excellent candidate for clinical trials in the treatment of cancer.

4-Demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548), a novel anticancer agent active against tumor cell lines with different resistance mechanisms.

The activity of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548), a new alkycycline with high antitumor activity against a broad range of cancer cells, was evaluated in vitro and in vivo in cells selected for resistance to different anticancer agents. Both in vitro and in vivo, PNU-159548 did retain its activity in cells expressing the multidrug resistance (MDR) phenotype, associated to MIDR-1 gene overexpression or with an alteration in the topoisomerase II gene (altered MDR), independently on the drug used for the selection of the resistant cell line. According to these data, the intracellular uptake of PNU-159548 is not influenced by the presence of MDR-1. PNU-159548 was also active, both in vitro and in vivo, against cells showing resistance to various alkylating agents iincluding cisplatin, cyclophosphamide, and melphalan) and topoisomerase I-inhibitors. Cells defective in nucleotide excision repair, which did show hypersensitivity to treatment with UV irradiation and alkylating agents, showed only a marginally increased sensitivity to PNU-159548. Similarly, the activity of the drug was not influenced by the mismatch repair system, as assessed in two different cellular systems deficient in hMLH1 expression and in which hMLH1 activity was restored by chromosome 3 transfer. The results obtained clearly indicate that the new anticancer agent PNU-159548 is able to overcome the classical mechanisms of resistance emerging after treatment with the most clinically used anticancer agents, and it could represent an alternate choice in the treatment of those tumors refractory to conventional therapy.

Modulation of the association reaction between hemoglobin and carbon monoxide by proton and chloride.

A cryogenic technique for the isolation of the ligation intermediates in the association reaction between hemoglobin and carbon monoxide at 20 degrees C [Perrella, M., Davids, N., and Rossi-Bernardi, L. (1992) J. Biol. Chem. 267, 8744-8751] was used to study the effects of proton and chloride concentrations on the rates of the stepwise reactions. The reaction rate was observed to increase continuously in the course of the ligation process, yet the acceleration of the reaction after the binding of two ligand molecules, observed previously in 100 mM KCl, pH 7, was not observed at other pH values. At pH 6.3, such an acceleration occurred after the binding of three ligands, and at pH 8.5, a large acceleration was observed after the binding of the first ligand molecule. Greater CO binding to the beta chains was observed under all conditions, as in the previous study. The functional heterogeneity of the chains in the first ligation step increased with pH. The chloride concentration did not influence the distribution of the ligand between the alpha and beta chains at pH 6.3 and 8.5. At pH 7, less binding to the alpha chains was observed at 7 mM chloride with respect to 100 mM. The nature of the biliganded component isolated at pH 7 in 100 mM KCl and unresolved by the cryogenic technique was studied using a combination of cryogenic and noncryogenic isoelectric focusing. This component was a mixture of intermediates (alpha beta) (alpha CO beta CO), about 65%, and (alpha beta CO) (alpha CO beta), about 35%. The experimental data were compared with the distributions of intermediates calculated according to the Monod kinetic model assuming rapid and concerted transitions between two quaternary structures at each ligation step. The model provided a qualitative fit of the observed distributions of intermediates at acidic and neutral pH. A large discrepancy between the experimental observations and the predictions of the model was found at alkaline pH. The mechanism of the association reaction is discussed in the light of the available information on the tertiary/quaternary structures of the intermediates, as obtained from the studies of the deoxy/cyanomet model of ligation.

Absorption and distribution of naproxen in rats orally treated with naproxen betainate sodium salt monohydrate. Comparison with naproxen.

The S-naproxen betainate sodium salt monohydrate (naproxen-betaNa, CAS 104124-26-7, Aprenin) was synthesized to improve bioavailability and tolerability of naproxen. 24 albino rats were treated with naproxen-betaNa (84 mg/kg) and 24 with S-naproxen (naproxen) (50 mg/kg) by the oral route, the doses being equimolar. The animals were sacrificed and naproxen was assayed in timed plasma samples drawn off over a 24-h period and in tissues excised 1 h after administration. Peak concentrations of naproxen proved to be higher with naproxen-betaNa than with naproxen as such. The area under the curve of naproxen concentrations observed with the two administrations overlapped as did concentrations of the drug in the lungs, myocardium and liver. Naproxen concentrations in the gastric wall after naproxen-betaNa proved to be lower than after administration of naproxen as such, which allowed the authors to assume that naproxen-betaNa has a better gastric tolerability.

Comparative bioavailability study on naproxen betainate sodium salt monohydrate and naproxen sodium salt in healthy volunteers.

The S-naproxen betainate sodium salt monohydrate (naproxen-betaNa, CAS 104124-26-7, Aprenin, test drug), and the sodium salt of S-naproxen (reference), were administered to twelve healthy volunteers of both sexes according to a crossover design, in a single dose of one 575 mg capsule of test, containing 342 mg of S-naproxen and two 275 mg tablets of reference, containing 502 mg of S-naproxen. Blood samples were drawn off over a 24-h period before (time 0) and after administration at foreseen time intervals. Naproxen was measured in plasma by a validated HPLC assay with UV detection which was able to detect 1 microgram/ml and proved to be linear in the range 1-100 micrograms/ml. The non-compartmental pharmacokinetic parameters obtained were statistically processed according to the EU guidance note on bioavailability and bioequivalence Cmax, AUC0-24h and AUC0-infinity were normalized to the dose of 502 mg of naproxen and log-transformed before statistical analysis to assess bioequivalence. Dose-normalized values of plasma concentrations encountered with the two formulations proved to overlap, with the exception of the first sampling time which showed naproxen concentrations that were higher with test drug than with reference. The specific test for bioequivalence led to 90% confidence intervals within the 80-125% range with target pharmacokinetic parameters, whereas the time to peak (tmax) observed with the test and reference drugs did not differ to any statistically significant degree when analysed with Wilcoxon's non-parametric test. It is concluded that the test drug should be declared bioequivalent with the reference drug in terms of dose-normalized concentrations, despite the more rapid increase in plasma concentrations of naproxen observed at the first sampling time with test drug.

Functional heterogeneity of the alpha and beta subunits in the association reaction between hemoglobin and carbon monoxide.

A technique is described for the rapid inactivation and removal of excess ferricyanide used for the non-cryogenic oxidation of the unliganded subunits of the intermediates in the association reaction between hemoglobin and carbon monoxide. Under these conditions the asymmetric oxidized intermediates, which dissociate into non-identical dimers, disproportionate into their parent tetramers and four species, Hb+, HbCO, alpha 2+ beta 2CO, alpha 2CO beta 2+, are isolated by non-cryogenic isoelectric focusing. The relative concentrations of species alpha 2CO beta 2+ and alpha 2+ beta 2CO measure the overall distribution of the ligand between the alpha and beta subunits in the association reaction. At 20 degrees C in 0.1 M KCl, pH 7, preferential CO binding to the beta subunits was observed, in agreement with observations made by the cryogenic technique for the isolation of the intermediates [M. Perrella, N. Davids and L. Rossi-Bernardi, J. Biol. Chem. 267 (1992) 8744].

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection.

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.

Stereospecific determination of amisulpride, a new benzamide derivative, in human plasma and urine by automated solid-phase extraction and liquid chromatography on a chiral column. application to pharmacokinetics.

Amisulpride, a drug belonging to the benzamide series, demonstrates antischizophrenic and antidepressant (antidysthymic) properties in man. For the pharmacokinetic studies of the racemic drug in man, a method of determination based on solid-phase extraction (SPE) from plasma and HPLC on a stereoselective column was developed. For this aim, one millilitre of plasma, after the addition of the internal standard, tiapride or metoclopramide, is diluted with a borate buffer at pH 9, then automatically loaded onto a SPE C18 100-mg column. The column is washed with different solvents, then eluted with 0.5 ml of methanol. After evaporation of the eluted fraction, the residue is reconstituted in 0.25 ml of eluent mixture. An aliquot is injected onto the HPLC column, a Chiralpak AS, equilibrated with an eluent mixture constituted by n-hexane-ethanol, (67:33, v/v) containing 0.2% (v/v) of diethylamine (DEA) or n-heptane-ethanol, (70:29.8, v/v) containing 0.2% of DEA and connected to a UV detector set at 280 nm or to a fluorimetric detector set at lambda ex = 280 nm and lambda cm = 370 nm. The limit of quantitation (LOQ) in human plasma is 2.5 ng ml-1 for both S-(-)- and R-(+)-amisulpride isomers with both detection methods. The method has been demonstrated to be linear in the range 2.5-320 ng ml-1 for both R-(+)- and S-(-)-amisulpride in human plasma with both UV and fluorescence detection. Absolute recovery of S-(-)- and R-(+)-amisulpride enantiomers from human plasma, as well as selectivity, precision and accuracy have been demonstrated to be satisfactory for pharmacokinetics in man and equivalent for both the proposed methods that have been cross-validated on real dosed human plasma samples. The methods have been used for clinical pharmacokinetic studies allowing pharmacokinetic parameters for amisulpride enantiomers in agreement with those obtained for the racemate to be obtained. After dilution with water, urinary samples from subjects treated with amisulpride racemate can be analysed according to the method used for plasma.