A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni.

A PCR-based method was developed for the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni (Xap), which provides rapid, sensitive and specific in planta detection and isolate identification. Primers specific for Xap were identified using random amplified polymorphic DNA (RAPD). Simplex PCR with these primers had a limit of detection per PCR reaction of approximately 10CFU for isolate cultures and 50CFU for plant material when used on tenfold dilutions of isolate culture or genomic DNA extracted from spiked samples, respectively. The primers were adapted as a high-throughput single-step screening based on a digoxigenin-labeled DNA probe assay with a detection limit of 4×10(2)CFU from isolate cultures. A duplex-PCR method was designed that includes the pathovar-level with species-level primers based on species-specific regions of the quinate metabolic gene qumA, increasing diagnostic confidence and offering the first molecular test for all X. arboricola pathovars.

A predictive model obtained by identification for the ultrasonic "equivalent" flow velocity at surface vicinity.

In the specific applications of surface cleaning and electrochemistry which consist of processes implanting surface irradiation by ultrasound, design of large-scale devices requires us to understand acoustic field distribution together with its quantification. This observation allows systematic measurement of ultrasonic stirring throughout electrochemical determination of "equivalent" flow velocity versus various operating parameters (powers, electrode-horn distances, reactor geometry, frequencies, etc.). A numerical model was proposed to fit our curves and to identify some parameters by taking into account the characteristics of the ultrasonic wave (absorption coefficient, rate of cavitation bubbles and acoustic power). Nevertheless, the flicked behavior of the ultrasonic processes in the vicinity of the electrode as well as bubble presence which induce non-linearities in wave propagation lead us to propose a new approach based on parameter identification by methods currently used in chemical engineering. These parameters were related to physical criteria, and the global model was evaluated throughout analysis of its sensibility criteria.

RPBS: a web resource for structural bioinformatics.

RPBS (Ressource Parisienne en Bioinformatique Structurale) is a resource dedicated primarily to structural bioinformatics. It is the result of a joint effort by several teams to set up an interface that offers original and powerful methods in the field. As an illustration, we focus here on three such methods uniquely available at RPBS: AUTOMAT for sequence databank scanning, YAKUSA for structure databank scanning and WLOOP for homology loop modelling. The RPBS server can be accessed at http://bioserv.rpbs.jussieu.fr/ and the specific services at http://bioserv.rpbs.jussieu.fr/SpecificServices.html.

Separation and identification of cannabis components by different planar chromatography techniques (TLC, AMD, OPLC).

The use of cannabis is illicit in numerous countries, and the increasing consumption has led to a multiplication of scientific studies. New methods of planar chromatography such as automated multiple development (AMD) and optimum performance laminar chromatography (OPLC) techniques can be used as a substitute for the traditional thin-layer chromatography for the identification and quantitation of the Indian hemp components. Each method offers its own advantage: high resolution with neither diffusion nor spot stretching for AMD and speed, efficiency, and the possibility of working in the semipreparative mode for OPLC.

OPLC and AMD, recent techniques of planar chromatography: their interest for separation and characterization of extractive and synthetic compounds.

It is always important for the chemist to have good methods of separation, characterization and quantitative evaluation for one or several compounds resulting from a chemical reaction or an extraction procedure. In this domain the chromatographic techniques are choice methods, in particular Over Pressured Layer Chromatography (OPLC) and Automated Multiple Development (AMD). They are relatively recent methods whose use is unfortunately not yet generalized although they give very clean separation. In this paper we present numerous examples of the use of these two new types of planar chromatographies and especially the results we have obtained in the field of natural products on a wide variety of different structures: coumarins, flavonoids, anthocyanins, alkaloids and essential oils.

Comparison of planar chromatographic methods (TLC, OPLC, AMD) applied to essential oils of wild thyme and seven chemotypes of thyme.

Essential oils analysis is more often realized by gas chromatography. However, thin-layer chromatography (TLC) remains the reference for Pharmacopoeia. Nevertheless classical TLC has its own limitations but it is always a good technique because it is simple, rapid and less expensive. Actually the reproducibility, the separation quality and the possibility to obtain good and reproducible quantitative determinations have been improved significantly with automated sample applicator, scanner densitometers and two new chromatographic planar methods: the optimum performance laminar chromatography (OPLC) and automated multiple development (AMD). In this work, we show and compare the performance of these methods and TLC through a study of seven thyme chemotypes and wild thyme essential oil.

[Atypical presentation of a carcinoid syndrome]. / Présentation atypique d'un syndrome carcinoïde

A 76-year-old woman presenting with right heart failure is reported. The investigation showed an ileal carcinoid tumour with multiple metastases and tricuspid regurgitation caused by a cardiac carcinoid lesion. Echocardiographic study and histopathological examination revealed typical characteristics of carcinoid heart disease. Nevertheless, except for the cardiac symptomatology, the patient did not show any other clinical manifestation of the carcinoid syndrome. Isolated right heart failure as the first sign of a carcinoid syndrome is rare; only two other cases have been described in the literature.

A comparative study of the effects of sparteine, lupanine and lupin extract on the central nervous system of the mouse.

Lupin is toxic because of its alkaloid content, sparteine and lupanine in particular. Although the pharmacological properties of sparteine are well known those of lupanine have not been much studied. This paper reports procedures for extraction, purification and crystallization of lupanine, and methods for the preparation of an extract for injection of Lupinus mutabilis Sweet, and for the determination of the acute toxicity and maximum non-lethal dose (DL0) of lupanine, sparteine and lupin extract in the mouse. The three substances were tested on the central nervous system (CNS) for locomotor activity, for interaction with specific drugs used for treatment of the CNS (the stimulant drugs amphetamine and pentetrazol and the depressant drugs pentobarbital and chlorpromazine) and for analgesic activity. The results indicate that lupanine and lupin extract are less toxic than sparteine and that at the doses studied the three products have a weak sedative effect on the CNS.

Pairwise and multiple identification of three-dimensional common substructures in proteins.

In this paper, we present an algorithm to find three-dimensional substructures common to two or more molecules. The basic algorithm is devoted to pairwise structural comparison. Given two sets of atomic coordinates, it finds the largest subsets of atoms which are "similar" in the sense that all internal distances are approximately conserved. The basic idea of the algorithm is to recursively build subsets of increasing sizes, combining two sets of size k to build a set of size k + 1. The algorithm can be used "as is" for small molecules or local parts of proteins (about 30 atoms). When a high number of atoms is involved, we use a two step procedure. First we look for common "local" fragments by using the previous algorithm, and then we gather these fragments by using a Branch and Bound technique. We also extend the basic algorithm to perform multiple comparisons, by using one of the structures as a reference point (pivot) to which all other structures are compared. The solution is the largest subsets of atoms common to the pivot and at least q other structures. Although both algorithms are theoretically exponential in the number of atoms, experiments performed on biological data and using realistic parameters show that the solution is obtained within a few minutes. Finally, an application to the determination of the structural core of seven globins is presented.

Automated multiple analysis of protein structures: application to homology modeling of cytochromes P450.

A computational strategy for homology modeling, using several protein structures comparison, is described. This strategy implies a formalized definition of structural blocks common to several protein structures, a new program to compare these structures simultaneously, and the use of consensus matrices to improve sequence alignment between the structurally known and target proteins. Applying this method to cytochromes P450 led to the definition of 15 substructures common to P450cam, P450BM3, and P450terp, and to proposing a 3D model of P450eryF.

P-SEA: a new efficient assignment of secondary structure from C alpha trace of proteins.

MOTIVATION: The secondary structure is a key element of architectural organization in proteins. Accurate assignment of the secondary structure elements (SSE) (helix, strand, coil) is an essential step for the analysis and modelling of protein structure. Various methods have been proposed to assign secondary structure. Comparative studies of their results have shown some of their drawbacks, pointing out the difficulties in the task of SSE assignment. RESULTS: We have designed a new automatic method, named P-SEA, to assign efficiently secondary structure from the sole C alpha position. Some advantages of the new algorithm are discussed. AVAILABILITY: The program P-SEA is available by anonymous ftp: ftp.lmcp.jussieu.fr directory: pub/.

Finding flexible patterns in a text: an application to three-dimensional molecular matching.

Finding certain regularities in a text is an important problem in many areas, e.g. in the analysis of biological molecules such as nucleic acids or proteins. In the latter case, the text may be sequences of amino acids or a linear coding of three-dimensional structures, and the regularities then correspond to lexical or structural motifs common to two, or more, proteins. We first recall an earlier algorithm that found these regularities in a flexible way. Then we introduce a generalized version of this algorithm designed for the particular case of protein three-dimensional structures, since these structures present a few peculiarities that make them computationally harder to process. Finally, we give some applications of our new algorithm on concrete examples.

About the large fluctuations observed using gas-phase molecular dynamics in the K-ras gene containing a mismatch.

We describe how we can reduce the periodic bending motions in the simulation in vacuo of the molecular dynamics of a short DNA fragment containing the Gly 12 hot spot of the K-ras oncogene and having at its center a mismatch CA+.

Comparison of the bis-intercalating complexes formed between either ditercalinium or a flexible analogue and d(CpGpCpG)2 or d(TpTpCpGpCpGpApA)2 minihelices: 1H- and 31P-NMR analyses.

The 400-MHz 1H- and 162-MHz 31P-nmr have been used to study complexes constituted by (a) the d(TpTpCpGpCpGpApA)2 or the d(CpGpCpG)2 self-complementary oligonucleotides and (b) two bifunctional 7H-pyrido [4,3-c] carbazole dimer drugs, the antitumoral ditercalinium (NSC 366241), a dimer with a rigid bis-piperidine linking chain and its pharmacologically inactive analogue, a dimer with a flexible spermine-like linking chain. Nearly all proton and phosphorus signals have been assigned by two-dimensional (2D) nmr (correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser enhancement spectroscopy, 2D 31P (1H) heteronuclear correlated spectroscopy and 31P-31P chemical exchange experiments). Both drugs bis-intercalate into the two CpG sites. The complexes show small differences in the position of the 7H-pyrido [4,3-c] carbazole ring into the intercalation site and possibly in the ribose-phosphate backbone deformation. However, the inactive analogue exhibits a longer residence lifetime in octanucleotide than the ditercalinium does. All these results are discussed in terms of differences in dimer activities.

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8.

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.

Reassessment of structural characteristics of the d(CGCG)2:actinomycin D complex from complete 1H and 31P NMR.

Complexes formed between Actinomycin D (ActD) and the tetranucleotides d(AGCT)2 and d(CGCG)2 were studied in detail by one and two-dimensional 1H and 31P NMR. The 31P two dimensional chemical exchange experiment, at room temperature on saturated complexes (1:1), showed unambiguously that the asymmetrical phenoxazone ring binds to the unique GC site under the two possible orientations in the d(AGCT)2 tetranucleotide but adopts a single orientation in the d(CGCG)2 tetranucleotide. For the d(CGCG)2:Act D saturated complex, complete assignments of all protons and phosphorus signals of the two-nucleotide strands, as well as of the two cyclic pentapeptide chains has allowed us to study in details the conformational features of the complex from NOE and coupling constants analysis. The tetranucleotide remains in a right-handed duplex, but the sugar puckers are modified for residues at the intercalation site. A uniform C2' endo pucker is observed for residues on the strand facing the quinoid side of the phenoxazone ring while a C2' endo-C3-endo equilibrium about 60% of C2' endo is proposed for the two residues on the strand facing the benzenoid side of the phenoxazone ring. In contrast to previous studies on ActD-DNA interactions, we have been able to measure the 3J phosphorus-proton coupling constants at the intercalation site but also adjacent to it, showing that 31P chemical shifts are not simply related to the backbone conformation. Molecular mechanics calculations, using empirical distances deduced from NOE effects as restrained distances during minimizations, led to a model differing mainly from those previously published by orientation of the N methyl groups of both N-Methyl-Valines.