Effect of the combination of several irrigants on dentine surface properties, adsorption of chlorhexidine and adhesion of microorganisms to dentine.

AIM: To investigate the effects of combinations of several irrigants on the roughness and wettability of dentine, adhesion of Enterococcus faecalis and Candida albicans and adsorption of chlorhexidine (CHX) to the dentine. METHODOLOGY: Bovine dentine samples were prepared and their surface roughness standardized. The samples were distributed in groups (n = 10) and subjected to one of the following irrigation protocols: G1 - saline solution; G2 - sodium hypochlorite (NaOCl); G3 - NaOCl + ethylenediaminetetraacetic acid (EDTA); G4 - NaOCl + peracetic acid (PAA); G5 - NaOCl + 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP); G6 - NaOCl + EDTA + CHX; G7 - NaOCl + PAA + CHX; G8 - NaOCl + HEDP + CHX; and G9 - mixture of NaOCl + HEDP. After treatments, roughness and wettability were measured. In order to evaluate the adhesion of microorganisms to dentine, new dentine samples were prepared and after 2 h of contact with the microorganisms, were analysed using a confocal laser scanning microscope and the number of microorganisms adhering to the surfaces were determined. Absorption spectra were collected by attenuated total reflectance of Fourier-transform infrared spectroscopy before and after immersion of other dentine samples in each solution of G6, G7 and G8 and in a solution of 2% CHX at various time intervals. The areas of the band associated with CHX with the peak at 1492 cm-1 were calculated between 1479 and 1500 cm-1 of the spectral range. The data obtained in all experiments were subjected to one-way ANOVA (α < 0.05). The values of the CHX band were also subjected to one-way repeated measures ANOVA (α < 0.05). RESULTS: Saline solution, NaOCl, HEDP and CHX did not alter the roughness of the dentine (P > 0.05), whilst EDTA and PAA did (P < 0.05). Dentine surface wettability increased after the use of all irrigants compared to saline solution (P < 0.05), with HEDP causing the greatest increases (P < 0.05). The adhesion of E. faecalis was favoured on surfaces treated with only saline solution and NaOCl, and on samples that had decalcifying agents as the final irrigant (P < 0.05). The adhesion of C. albicans was highest on surfaces treated with only saline solution and on surfaces that had NaOCl used as the last irrigant (P < 0.05). The use of CHX as a final irrigant reduced the adhesion of both microorganisms. The roughness and wettability did not influence the adhesion of the microorganisms tested. The adsorption of CHX to the dentine was significant after 1 min of immersion of the mineralized samples in the irrigant (P < 0.05), and the use of chelating agents prior to CHX potentiated this adsorption. CONCLUSIONS: The irrigation solutions had a variable effect on the properties of dentine, on the adhesion of E. faecalis and C. albicans and the adsorption of CHX to the dentine surface.

Possible drug-drug interaction between high-dose esomeprazole and phenprocoumon.

PURPOSE: It is established that omeprazole increases (R)+ warfarin levels with around 10 %. Whether (es)omeprazole also increase the plasma levels of acenocoumarol or phenprocoumon is still uncertain. We analyzed whether addition of (es)omeprazole to acenocoumarol or phenprocoumon increases the international normalized ratio (INR) levels and the risk of overanticoagulation. METHODS: We analyzed all hospital admissions in four teaching hospitals. Patients who used coumarins and pantoprazole or (es)omeprazole simultaneously for at least four consecutive days were included in the study. We analyzed the highest INR level and whether patients had an INR level above six. We compared patients using omeprazole or esomeprazole with patients using pantoprazole, because for pantoprazole, no interaction has been reported. RESULTS: We analyzed 5747 admissions with 4540 patients using one of the drug combinations. For acenocoumarol (4578 admissions), no significant differences were found between users of esomeprazole, omeprazole, and pantoprazole. For phenprocoumon (1169 admissions), the highest INR measured was significantly higher in users of esomeprazole than in users of pantoprazole (4.7 versus 4.3; p = 0.035). No significant difference was found with omeprazole versus pantoprazole (4.3 versus 4.3; p = 0.66). A non-significant association was found between the esomeprazole dose and the highest INR level (p = 0.055). The risk of an INR above six did not differ significantly between esomeprazole and pantoprazole (27.7 % versus 22.9 %; p = 0.34). CONCLUSIONS: The use of esomeprazole simultaneously with phenprocoumon during hospital admissions might increase the anticoagulant effect. The clinical relevance seems to be limited, because no statistically significant increased risk of overanticoagulation was found.

A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions.

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

Liquid chromatography-mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection.

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with liquid chromatography-mass spectrometry (LC-MS), for the analysis of biological samples is described. The system was tested with cortisol and prednisolone for plasma analysis and arachidonic acid for urine analysis. A precolumn packed with a 25-micron C18 alkyl-diol support is used for direct plasma or urine injection. Using column-switching techniques, the analytes enriched on the precolumn are eluted to the analytical column without transfer loss. An on-line heart-cut technique was employed and only the analyte-containing fraction eluting from the LC column is directed to the MS to protect the LC-MS interface and ion-source from contamination. The whole system is operated in a parallel mode, that is, sample pre-treatment and LC-MS analysis are performed simultaneously to provide the shortest possible analysis time. The only off-line sample pre-treatment step required was centrifugation to remove particulate matter. With the fully automated system, total analysis times of 5 and 9.5 min were achieved for cortisol in serum and arachidonic acid in urine, respectively. Cortisol and related compounds were quantitatively recovered from plasma with a detection limit for prednisolone (direct injection of 100 microliters on restricted-access precolumn) of 2 ng/ml.

Energetic limits to metabolic flexibility: responses of Saccharomyces cerevisiae to glucose-galactose transitions.

Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose-galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose-galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.

Characteristics of the tomato nuclear genome as determined by sequencing undermethylated EcoRI digested fragments.

A collection of 9,990 single-pass nuclear genomic sequences, corresponding to 5 Mb of tomato DNA, were obtained using methylation filtration (MF) strategy and reduced to 7,053 unique undermethylated genomic islands (UGIs) distributed as follows: (1) 59% non-coding sequences, (2) 28% coding sequences, (3) 12% transposons-96% of which are class I retroelements, and (4) 1% organellar sequences integrated into the nuclear genome over the past approximately 100 million years. A more detailed analysis of coding UGIs indicates that the unmethylated portion of tomato genes extends as far as 676 bp upstream and 766 bp downstream of coding regions with an average of 174 and 171 bp, respectively. Based on the analysis of the UGI copy distribution, the undermethylated portion of the tomato genome is determined to account for the majority of the unmethylated genes in the genome and is estimated to constitute 61+/-15 Mb of DNA (approximately 5% of the entire genome)--which is significantly less than the 220 Mb estimated for gene-rich euchromatic arms of the tomato genome. This result indicates that, while most genes reside in the euchromatin, a significant portion of euchromatin is methylated in the intergenic spacer regions. Implications of the results for sequencing the genome of tomato and other solanaceous species are discussed.

Biosynthesis and elongation of short- and medium-chain-length fatty acids.

Short- and medium-chain-length fatty acids (FAs) are important constituents of a wide array of natural products. Branched and straight short-chain-length FAs originate from branched chain amino acid metabolism, and serve as primers for elongation in FA synthase-like reactions. However, a recent model proposes that the one-carbon extension reactions that utilize 2-oxo-3-methylbutyric acid in leucine biosynthesis also catalyze a repetitive one-carbon elongation of short-chain primers to medium-chain-length FAs. The existence of such a mechanism would require a novel form of regulation to control carbon flux between amino acid and FA biosynthesis. A critical re-analysis of the data used to support this pathway fails to support the hypothesis for FA elongation by one-carbon extension cycles of alpha-ketoacids. Therefore, we tested the hypothesis experimentally using criteria that distinguish between one- and two-carbon elongation mechanisms: (a) isotopomer patterns in terminal carbon atom pairs of branched and straight FAs resulting from differential labeling with [(13)C]acetate; (b)(13)C]threonine labeling patterns in odd- and even chain length FAs; and (c) differential sensitivity of elongation reactions to inhibition by cerulenin. All three criteria indicated that biosynthesis of medium-chain length FAs is mediated primarily by FA synthase-like reactions.

Thermospray liquid chromatography/mass spectrometry of polar beta-blocking drugs: preliminary results.

The use of thermospray liquid chromatography for the screening of polar beta-blocking drugs is evaluated. The influence of instrumental parameters on the fragmentation pattern of labetalol, acebutolol, diacetolol, sotalol and atenolol is described. A short biomedical application is presented.

Comparative bioavailability study of two haloperidol decanoate containing products.

In our hospital pharmacy an injectable solution of haloperidol decanoate 141 mg/ml (equivalent to haloperidol 100 mg/ml) in sesame oil was prepared. The aim of this study was to prove bioequivalence of this formulation with the reference product, Haldol Decanoas. 15 schizophrenic patients, already stabilized with Haldol Decanoas, were enrolled. Intramuscular injections were given every three weeks in the following doses: 100 mg (1 x), 200 mg (7 x) and 300 mg (7 x). In this open, randomized, cross-over study all patients received four injections of the reference product, and four injections of the test product. Only after the fourth injection of each product (when steady-state levels were reached) a concentration-time profile of haloperidol was established during the dose interval of 21 days. The pharmacokinetic parameters AUC0-21 and Cmax were statistically evaluated. Based on these parameters the conclusion was drawn that both products were bioequivalent. The preparation of this injectable haloperidol decanoate solution in our hospital pharmacy amounts to an annual saving of approximately $39,000.

On-line liquid chromatography/thermospray mass spectrometry in the analysis of oligosaccharides.

The analysis of intact neutral oligosaccharides by on-line liquid chromatography/thermospray mass spectrometry is described. Molecular-weight information on oligomers up to a degree of polymerization of 10 is obtained using an aqueous mobile phase containing 10(-4) mol/L sodium acetate, which was found to be compatible with thermospray interfacing and ionization. Ions due to sodiated and disodiated oligosaccharides are observed under these conditions without fragmentation. The aqueous 10(-4) mol/L sodium acetate mobile phase is demonstrated to be applicable in the separation of mixtures of oligosaccharides on a reversed-phase octadecyl-modified silica column.

Genetic control and evolution of sesquiterpene biosynthesis in Lycopersicon esculentum and L. hirsutum.

Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase1 (Sst1), was identified for which the L. esculentum allele is associated with the biosynthesis of beta-caryophyllene and alpha-humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B, and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6-associated sesquiterpenes. At a second locus, Sst2, on chromosome 8, the L. hirsutum allele specified accumulation of alpha-santalene, alpha-bergamotene, and beta-bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.

Structure elucidation of mitomycin C metabolites. I. The applicability of various mass spectrometric techniques in metabolic studies on mitomycin C.

Knowledge of the metabolism of the anticancer drug mitomycin C (MMC) is necessary in order to optimize the administration schemes of this drug. The present study concentrates on the metabolism of MMC in liver homogenates. Various mass spectrometric methods have been investigated for their usefulness in the metabolic studies on MMC by comparing the mass spectral data. Data from electron impact, direct probe chemical ionization, direct chemical ionization, field desorption and thermospray ionization have been obtained. One of the soft ionization methods, i.e. direct chemical ionization, has been applied to the detection of MMC in a spiked piglet liver sample. It appears to be necessary to enhance the selectivity of the method by using tandem mass spectrometry, owing to the high biological background.

Liquid chromatography-thermospray tandem mass spectrometry for identification of a heptabarbital metabolite and sample work-up artefacts.

An unknown heptabarbital metabolite, observed in the liquid chromatogram of rat plasma and urine samples after administration of heptabarbital, was identified by liquid chromatography-thermospray tandem mass spectrometry. By applying the parent scan mode for screening and the daughter scan mode for structure elucidation, the metabolite was determined to be 5-ethyl-5-(1'-, 3'- or 6'-cycloheptadienyl)barbituric acid. It was demonstrated that artefact formation occurred when hydrochloric acid was used for conjugate hydrolysis in the sample clean-up. Identification of the artefacts was achieved by the same method. Confirmation of the structures of the metabolite and the artefacts was obtained by gas chromatography-electron impact mass spectrometry.

On-line continuous-flow dialysis thermospray tandem mass spectrometry for quantitative screening of drugs in plasma: rogletimide.

The application of a continuous-flow dialysis system, consisting of a membrane dialyser and a trace enrichment column, in on-line combination with tandem mass spectrometry via a thermospray interface is described. The method is applied to the quantitation of drugs in complex biological matrices containing macromolecular interferences. The potential of the method is demonstrated by the quantitative analysis of the anti-cancer drug rogletimide in the plasma of patients after treatment.

Automated analysis of mitomycin C in body fluids by high-performance liquid chromatography with on-line sample pre-treatment.

A fully automated liquid chromatographic system for the bioanalysis of mitomycin C has been described. The isolation of the analyte from the biological matrix (plasma, ascites and urine) is performed using a continuous-flow system equipped with a dialysis membrane in order to remove proteins. The samples are concentrated on a reversed-phase pre-column and subsequently introduced on to a reversed-phase analytical column by applying column-switching techniques. The drug is detected by absorbance measurements at 360 nm. Using the described system up to 100 samples a day can be analysed with determination limits of the order of 1 ng/ml, with a linear dynamic range of at least three decades for plasma and urine samples. The procedure was applied to pharmacokinetic studies of ovarian cancer patients treated intraperitoneally with mitomycin C.

Identification of 4-hydroxyandrost-4-ene-3,17-dione metabolites in prostatic cancer patients by liquid chromatography-mass spectrometry.

Liquid chromatography with thermospray mass spectrometry has proved to be an invaluable technique for the study of metabolic degradation of xenobiotics in complex biological fluids. This paper describes the detection of 4-hydroxyandrost-4-ene-3,17-dione and its metabolites in urinary extracts from prostatic cancer patients. Several metabolites were detected including 4 beta,5 alpha-dihydroxyandrostan-3,17-dione, 3,17-dihydroxyandrostan-4-ones and 3 alpha-hydroxy-5 beta-androstan-4,17-dione.

Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development.

Using a highly synchronous in vitro tuberization system, in combination with an amplified restriction fragment polymorphism (AFLP)-derived technique for RNA fingerprinting (cDNA-AFLP), transcriptional changes at and around the time point of potato tuberization have been analyzed. The targeted expression analysis of a specific transcript coding for the major potato storage protein, patatin and a second transcript, coding for ADP-glucose pyrophosphorylase, a key gene in the starch biosynthetic pathway is described. This paper confirms that kinetics of expression revealed by cDNA-AFLP analysis are comparable to those found in Northern analysis. Furthermore, this paper reports the isolation and analysis of two tuber-specific transcript-derived fragments (TDFs) coding for the lipoxygenase enzyme, which are differentially induced around the time point of tuber formation. Analysis of the two lox TDFs demonstrates that it is possible to dissect the expression modalities of individual transcripts, not independently detectable by Northern analysis. Finally, it is shown that using cDNA-AFLP, rapid and simple verification of band identity may be achieved. The results indicate that cDNA-AFLP is a broadly applicable technology for identifying developmentally regulated genes.

Gradient reversed-phase liquid chromatography coupled on-line to receptor-affinity detection based on the urokinase receptor.

A postcolumn receptor-affinity detection (RAD) was developed for the detection of urokinase and cross-reactive compounds. The analytical method consisted of gradient reversed-phase HPLC coupled on-line to a RAD system based on fluorescein-labelled urokinase receptor (fluorescein-uPAR) as reagent. Fluorescein-uPAR was added continuously to the HPLC effluent to react with analytes eluting from the LC column. Unreacted fluorescein-uPAR was removed by a short affinity column packed with an immobilised urokinase support. The analyte-bound fluorescein-uPAR fraction passes the affinity column unretained and was detected downstream by means of a fluorescence detector. An absolute detection limit of 40 fmol urokinase was obtained in the flow injection mode. In the gradient HPLC-RAD system a detection limit of 40 nM (20-microl injection, absolute amount, 800 fmol) was obtained. The present method allowed the identification of active breakdown products of urokinase both in standard samples and biological matrices.

Pharmacokinetics and pharmacodynamics of midazolam administered as a concentrated intranasal spray. A study in healthy volunteers.

AIMS: To investigate the pharmacokinetic and pharmacodynamic profile of midazolam administered as a concentrated intranasal spray, compared with intravenous midazolam, in healthy adult subjects. METHODS: Subjects were administered single doses of 5 mg midazolam intranasally and intravenously in a cross-over design with washout period of 1 week. The total plasma concentrations of midazolam and the metabolite 1-hydroxymidazolam after both intranasal and intravenous administration were described with a single pharmacokinetic model. beta-band EEG activity was recorded and related to midazolam plasma concentrations using an exponential pharmacokinetic/pharmacodynamic model. RESULTS: Administration of the intranasal spray led to some degree of temporary irritation in all six subjects, who nevertheless found intranasal administration acceptable and not painful. The mean (+/-s.d.) peak plasma concentration of midazolam of 71 (+/-25 ng ml-1) was reached after 14 (+/-5 min). Mean bioavailability following intranasal administration was 0.83+/-0.19. After intravenous and intranasal administration, the pharmacokinetic estimates of midazolam were: mean volume of distribution at steady state 1.11+/-0.25 l kg-1, mean systemic clearance 16.1+/-4.1 ml min-1 kg-1 and harmonic mean initial and terminal half lives 8.4+/-2.4 and 79+/-30 min, respectively. Formation of the 1-hydroxymetabolite after intranasal administration did not exceed that after intravenous administration. CONCLUSIONS: In this study in healthy volunteers a concentrated midazolam nasal spray was easily administered and well tolerated. No serious complications of the mode of administration or the drug itself were reported. Rapid uptake and high bioavailability were demonstrated. The potential of midazolam given via a nasal spray in the acute treatment of status epilepticus and other seizure disruptions should be evaluated.