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.

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.

Ricin A chain conjugated with monoclonal antibodies selectively killing human carcinoma cells in vitro.

Ricin A chain was coupled to murine monoclonal antibodies MBr1 and MOv2 respectively raised against human breast and ovarian carcinomas. Inhibition of protein synthesis only occurred in those cultured human tumor cells bearing the appropriate target antigens, demonstrating that both components of the conjugate were unchanged in regards to specificity and toxicity. Conjugates were 125-200 times more efficient in inhibiting [3H]proline incorporation than the uncoupled ricin A chain. They were however unable to kill the entire population of the appropriate cells even after repeated treatment. Although the two monoclonal antibodies had similar binding kinetics, the conjugates differed in their cytotoxicity kinetics. The MBr1-ricin A chain conjugate had slow kinetics, and about 20 hours were needed to obtain a protein synthesis inhibition above 50% on the appropriate line (mammary carcinoma MCF-7). In contrast, the MOv2-ricin A chain conjugate showed very fast kinetics, reaching 50% inhibition after only 30 minutes of treatment on both appropriate cell lines SW626 and HT-29 from ovarian and colon carcinomas, respectively. Growth conditions of cell lines, i.e., adherent cells versus suspended cells, and plating time were found to greatly influence the conjugates' killing efficiencies. These studies confirm the possibility of preparing ricin A chain-antibody conjugates, which retain specific cytotoxicity against tumor cells; but they also underline the need for further in vitro studies of various parameters before one considers a therapeutic use of such conjugates.

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.

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.

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].

What the intermediate compounds in ligand binding to hemoglobin tell about the mechanism of cooperativity.

The populations of the intermediates in concentrated solutions of hemoglobin A0 equilibrated at various PCO values, pH 7.0, 0.1 M KCl, and 20 degrees C, have been determined using cryogenic methods. Data on CO saturations and distributions of intermediates were analysed in terms of the free energies of dimer-tetramer assembly of the intermediates (G.K. Ackers and F.R. Smith, Annu. Rev. Biophys. Chem. 16 (1987) 583). The cooperative free energy value of the singly ligated species was approximately one-half the total cooperative energy. The cooperative free energy value of the doubly ligated species was not significantly different from that of carboxyhemoglobin. Because of experimental error, the observed difference in concentrations among the populations of the doubly ligated species cannot be taken as indicative of their functional heterogeneity. Additional studies on some NO intermediates have emphasized that (alpha 1 beta 1)(alpha 2 beta 2)X, a key intermediate in the formulation of the 'third-state' hypothesis in the deoxy/cyanomethemoglobin system, has a free energy value for dimer-tetramer assembly which is critically dependent on the nature of the ligand X as suggested by Ackers and Smith (reference as cited above).

Determination of alpidem, an imidazopyridine anxiolytic, and its metabolites by column-switching high-performance liquid chromatography with fluorescence detection.

Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo[1,2-a]pyridine- 3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of plasma samples (200 microliters) on to a precolumn filled with C18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the precolumn was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C8 type and the mobile phase was acetonitrile-methanol-phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 ml min-1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng ml-1, respectively, in human plasma.

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.

Monoclonal anti-CEA antibody: factors affecting localization in a preclinical study.

131I-labelled anti-CEA monoclonal antibody was tested in an animal model to evaluate: influence of antibody type (whole versus F(ab')2 fragments), administration route (i.v. versus i.p.), dose of tracer (100 microCi versus 1000 microCi), growth site (s.c. versus i.p.) and size of tumor. Athymic mice bearing CEA-producing human colon carcinoma (HT-29) or human melanoma as an irrelevant tumor (MeWo) received tracer and immunoscintigraphy and the localization ratios (LR) were compared. In HT-29 bearing animals F(ab')2 fragments localized better than the whole antibody. The LR were higher after i.p. administration of the tracer, independently of the tumor characteristics or the injected dose. The highest values were achieved when the radioactivity remaining in the whole body was below 2% of the injected dose. The images were negative when the i.p. injected dose was low or tumor growth was i.p. but positive in the other conditions (i.v. administration, high tracer dose, s.c. tumor growth). In the animals bearing melanoma, images scored positive or negative when the tumor weight was respectively above or below 400 mg, but the LR were always low.

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.7) and plasma cholinesterase (EC 3.1.1.8) activity, based on the measurements of pH variation due to release of acetic acid from acetylcholine. The major advantages of the differential pH procedure are simplicity, high reproducibility, no need for pre-treatment of samples, automatic correction of sample blanks, and speed and direct measurement of enzymatic reaction. The proposed methods are linear up to 7400 U/L at 30 degrees C and correlate well with the manual spectrophotometric method of Ellman for plasma cholinesterase and for washed erythrocytes. We adapted the same technique for the determination of erythrocyte cholinesterase using whole blood as sample and quinidine sulphate as inhibitor of pseudocholinesterase.

New anthracycline glycosides from Micromonospora. II. isolation, characterization and biological properties.

Four glycosides, designated A, B, C and D, are the main components of the anthracycline complex produced by cultures of Micromonospora sp. nov. They were extracted by solvent partition, separated by column chromatography and characterized by chemical and physical methods as 11-deoxy analogues of daunorubicin. Among these new anthracyclines, displaying antibacterial and cytotoxic activity in vitro, 11-deoxydaunorubicin and 11-deoxydoxorubicin are also active against P388 leukemia in mice.

The effect of human serum on the binding activity of radiolabelled monoclonal antibodies.

Three murine monoclonal antibodies (MoAbs), MBr1 and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabelled MoAbs were analyzed for their "in vitro" stability in human blood. They were incubated at 37 degrees C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

Urea determination in dialysis, based on a differential pH technique.

The application of a new technique, based on differential measurements of pH, to determine urea concentration in patients of a dialysis center, is reported. Urea in plasma, whole blood or dialysis fluids is measured by an enzymatic reaction, with urease; the procedure, requiring 10 microL of sample, is simple, fast and correlates well with a reference spectrophotometric method, in the 0-300 mg/dL concentration range, according to the equation y = 1.0291 X -0.0777; r = 0.9991; n = 73.

Carbohydrate epitope defined by an antitumor monoclonal antibody detected on glycoproteins and a glycolipid by immunoblotting.

The murine monoclonal antibody (MAb) MOv2 was found to be directed against the carbohydrate moieties of different kinds of molecules expressed on a human ovarian cystoadenocarcinoma. To define further the glycoconjugates carrying the MOv2-defined epitope, different procedures were used to analyze materials from surgical specimens and carcinoma cell lines. SDS-PAGE and immunoblotting showed glycoprotein molecules migrating in the gel as high and intermediate molecular weight components. A low-molecular-weight band, migrating approximately with the dye front, was also immunostained by MOv2. On the other hand, the immunostaining of high-performance thin-layer chromatography (HPTLC) of the total glycolipid extract and its neutral and acid fractions, after DEAE chromatography, showed selective reactivity with a neutral glycolipid. Reanalysis by immunoblotting of this glycolipid band scraped off the HPTLC plate indicated that it corresponds to the low-molecular-weight component. Periodate oxidation and Pronase digestion further demonstrate the saccharide nature of the determinant on both types of glycoconjugates. In conclusion, we report evidence that with a single analytical procedure, i.e., immunoblotting, it is possible to recognize the same carbohydrate determinant carried on both protein and lipid molecules.

Change in binding reactivity of an anti-tumor monoclonal antibody after the introduction of 2-pyridyl disulphide groups.

The use of monoclonal antibodies for in vivo therapeutic approaches depends largely on their specificity. During the characterization of ricin A-chain-murine monoclonal antibody conjugates we found that the binding specificity of a monoclonal antibody raised against human ovarian carcinoma (MOv2) seemed altered. Therefore, the binding reactivity of the unmodified antibody (MOv2), the conjugation intermediate (MOv2-PDP) and the conjugate (MOv2-A chain) was titrated by solid-phase radioimmunoassay on 11 human tumor cell lines belonging to seven different histotypes. The three reagents bound with the two reference cell lines (SW626:ovary carcinoma and HT-29:colon carcinoma). The MOv2-PDP and the Mov2-A chain also reacted with seven other cell lines which were unreactive with the unmodified MOv2. In addition MOv2-PDP exhibited reactivity on all normal cells tested (peripheral blood lymphocytes and skin fibroblasts). To elucidate the significance of these findings the following experiments were performed: cross inhibitions between the unmodified and modified monoclonal antibodies; comparative absorption tests with different cell lines; and immunoblotting analysis of the target antigens. The results suggest that after chemical modification with SPDP the monoclonal antibody MOv2 increases its binding activity, so that even a low number of antigenic sites can be detected. This study underlines the need to redefine the specificity of a conjugate before considering therapeutic applications.

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.

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.