Orwik

Quantification of dichromatism: a characteristic of color in transparent materials.

The color of a material, such as solution of a dye, can change by changing parameters like pH, temperature, illumination direction, and illumination type. Dichromatism -- a color change due to the difference in thickness of the material -- has long b... expand abstracteen known as a property of only a few materials. Here we show that dichromatism is a common property of many substances and materials, and we introduce a method for its quantification. We defined dichromaticity index (DI) as the difference in hue angle (Delta h(ab)) between the color of the sample at the dilution, where the chroma is maximal, and the color of four times more diluted (or thinner) and four times more concentrated (or thicker) sample. The two hue angle differences are called dichromaticity index toward lighter (DI(L)) and dichromaticity index toward darker (DI(D)), respectively. High dichromaticity was found for materials that were previously known as dichromatic (pumpkin oil, bromophenol). collapse abstract

Screening of selected food and medicinal plant extracts for pancreatic lipase inhibition.

Lipids are important components in human nutrition; however, their increased intake contributes to the development of obesity and can lead to multiple long-term complications. Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is a key enz... expand abstractyme for the absorption of dietary triglycerides. Interference with fat hydrolysis results in the reduced utilization of ingested lipids, therefore inhibition of lipases decreases fat absorption. Extracts from 106 species of medicinal plants, vegetables and fruits were screened for potential lipase inhibitory activity. p-Nitrophenylpalmitate and 5-bromo-4-chloro-3-indoxylpalmitate were used as substrates in an in vitro test with crude porcine pancreatic lipase. Bearberry (Arctostaphylos uva-ursi), garden pea (Pisum sativum), Norway spruce (Picea abies) and large-leaved lime (Tilia platyphyllos) extracts were the most active. Additionally, the activity of selected extracts with removed polyphenols was measured. Extracts of bearberry, garden pea and large-leaved lime are a promising source for developing functional foods or isolating active compounds. collapse abstract

The impact of total control of antibiotic prescribing by infectious disease specialist on antibiotic consumption and cost.

We analyzed the impact on antimicrobial consumption of antimicrobial therapy totally guided by an infectious disease specialist (iD). in a teaching hospital antibiotic consumption was recorded yearly from 1998 to 2005 in three units. Antibiotic polic... expand abstracty was introduced in 1999. in Unit A, restricted antimicrobials were prescribed after approval by the head of the unit. the iD was involved as a consultant upon request. in unit b, restricted antimicrobials were approved by an iD. All other antimicrobials were prescribed by the physicians on the ward. in unit C, all antimicrobials were prescribed by an iD. A significant decreasing trend in antibiotic consumption in defined daily doses (DDD) per patient day and per admission, and cost of antimicrobials per patient was observed in unit C, a decreasing trend in antibiotic consumption per patient was observed in unit b as well. totally iD guided antimicrobial therapy is an efficient method of antimicrobial policy. collapse abstract

Membrane permeability of acylated cystatin depends on the fatty acyl chain length.

Hydrophobization of proteins, such as chemical acylation, has been recognized as an efficient method for improving their membrane permeability. In this research, chicken cystatin, a model protein inhibitor of cysteine proteinases, was acylated with f... expand abstractatty acyl residues of 6-18 carbon atoms. The chemical modification was performed using fatty acyl chloride dispersion in aqueous acetonitrile solution. The reaction products were analyzed by capillary electrophoresis, SDS-PAGE and isoelectric focusing. In vitro inhibitory activity was determined by N-benzoyl-D,L-arginine-beta-naphthylamide assay and membrane permeability properties of non-acylated and acylated cystatin by measuring its efficiency to inhibit intracellular cathepsin B in MCF-10A neo T cells. The experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin B. In contrast, non-acylated cystatin did not cause inhibition as it was unable to enter the cell. The permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with stearoyl chloride. In addition, chemical modification did not influence the protein's immunogenicity. collapse abstract

Ultrasound in phage display: a new approach to nonspecific elution.

Libraries of phage-displayed random peptides are routinely used to identify target-binding peptides. Phages are commonly eluted in a nonspecific manner, especially if there are no available ligands of the particular target to use as competitors. Howe... expand abstractver, the present study clearly demonstrates that nonspecific elution is not always able to break peptide-target interactions. To circumvent this we have developed an improved nonspecific elution strategy that uses ultrasound to release target-bound phages and enables selection of high-affinity clones in a single step. collapse abstract

Identification of herbarium whole-leaf samples of Epilobium species by ATR-IR spectroscopy.

A simple, high-accuracy FT-IR method based on attenuated total reflection (ATR) was developed for the rapid determination of leaf samples of Epilobium species. The method is superior to other analytical techniques, since there is no need of laborious... expand abstract sample preparation such as grinding or extraction and solvent removal. A total of 70 herbarium specimens, belonging to all 13 Epilobium and to 2 Chamerion species growing in Slovenia, were analyzed. With the 100 most-informative wavenumbers in the range 700-1800 cm(-1), we obtained over 90% accuracy of species identification, with discriminant multivariate statistical analysis on the measurements made on whole dried leaves. collapse abstract

Comparison of capillary electrophoresis and high performance liquid chromatography for determination of flavonoids in Achillea millefolium.

Flavonoids represent an important bioactive component in Achillea millefolium. The comparison of the most commonly used analytical methods for the identification and quantification of flavonoids, capillary electrophoresis (CE) and high performance li... expand abstractquid chromatography (HPLC), is presented. The methods were optimized and validated. Using a 20 mM borate buffer with 30% (v/v) of methanol (pH 9.3) in the CE analysis and a gradient elution with water-acetonitrile mobile phase in the HPLC analysis, sufficient separation of the analytes was achieved. A relatively high injection volume in the CE analysis (30 mbar x 30s) enabled low limit of detection (LOD) (0.3-0.7 mg/L). Repeatability of both methods was acceptable (relative standard deviation of peak area were <6%). Additionally, the amount of flavonoids in a real sample of the dried herbal drug was determined. collapse abstract

Physicochemical and physiological basis of dichromatic colour.

Out of three perceptual characteristics of the colour of any substance, the hue depends mostly on the spectral properties of a substance, while the brightness and saturation depend also on the concentration of a substance and its thickness. Here, we ... expand abstractreport that evident change of the hue of the colour (i.e., from green to red) is due to a change in concentration or the thickness of a layer in some exceptional substances such as pumpkin seed oil or an aqueous solution of bromophenol blue. In some regions of Central Europe, salad dressing is made preferably with the pumpkin seed oil, which has a strong characteristic nut-like taste and remarkable properties of the colour: it appears red in a bottle, but green when served as a salad dressing. The colour of the pumpkin seed oil was previously described as brownish yellow, dark green, dark green to red ochre or dark reddish brown to light yellow green. We elucidated the physicochemical and physiological basis of such dichromatism by Beer-Lambert law and by the characteristics of human colour perception. Our concept was corroborated by the outcome of calculations of colour from spectral properties using colour matching functions. We found that dichromatism is observed if the absorption spectrum of any substance has at least two local minima: one wide but shallow and one narrow but deep local minimum. collapse abstract

Precision of micellar electrokinetic capillary chromatography in the determination of seven antibiotics in pharmaceuticals and feedstuffs.

Validation of analytical procedures is important for their efficient and reliable application. The International Conference on Harmonisation (ICH), Food and Drug Administration (FDA) and pharmacopoeia guidelines achieved a great deal in harmonising t... expand abstracthe definitions of the required validation characteristics. It is well known that poor reproducibility limits the practical implementation of capillary electrophoresis (CE). A precision study on four different MEKC methods was performed with 11 samples, containing seven antibiotics, by two analysts, in few days, on two capillary electrophoresis instruments. Five pharmaceutical preparations and three animal feeds were used. Precision was statistically analysed using migration time, peak area and height of each compound, as well as electroosmotic front (EOF). In 25 of 31 cases, the reproducibility of peak area, peak height and migration time was good (<5%). In most cases the reproducibility of peak area was much better than the reproducibility of peak height. The worst reproducibility that we observed was 12.7% for peak height and 7.6% for peak area. collapse abstract

Determination of xanthohumol in hops (Humulus lupulus L.) by nonaqueous CE.

Xanthohumol (XN) is a prenylated chalcone with antimutagenic and anticancer activity from hops. A nonaqueous reverse polarity capillary electrophoretic method for the determination of XN in hop extract was developed and validated. The optimal paramet... expand abstracters were a 64.5 cm long fused-silica capillary with 50 microm id at 25 degrees C; 30 kV negative voltage (anode at detector side of the capillary); nonaqueous buffer with 75 mM NaOH and 50 mM boric acid in methanol; hydrodynamical injection with 10 mbar for 40 s; and detection at 440 nm. XN, isoxanthohumol (IX), colupulone, adlupulone, and n-lupulone were well resolved on the electropherogram. The LOD for XN was 0.05 mg/L and RSD for peak area was below 3%. The amount of XN in different samples of hop pellets varied from 0.14 to 0.42%. collapse abstract

A new method for the authentication of plant samples by analyzing fingerprint chromatograms.

Chemical analysis by high-performance liquid chromatography or capillary electrophoresis of plant pulverized samples, juices or extracts is an excellent method for the authentication of medicinal plant species and their products, particularly when mo... expand abstractrphological authentication is not possible. In the conventional procedure, chromatograms are integrated and the heights or areas of several peaks are used in a supervised pattern recognition method to confirm the authenticity of the product. We propose a new section approach in analysing chromatograms, where chromatograms are split into sections, which are described by four variables (number of peaks in the section, average retention time of peaks in the section, total area of peaks in the section and average area of peaks in the section), and these variables are then used in statistical analysis. The method is especially useful when the peaks on the chromatogram are not well separated and it is not easy to link individual peaks on one chromatogram with corresponding peaks on other chromatograms. In comparison with the standard procedure, our approach in analyzing chromatographic data of willow-herb (Epilobium and Chamaenerion spp.) extracts was more objective, gave better results and was also easier to perform. collapse abstract

Improved acylation method enables efficient delivery of functional palmitoylated cystatin into epithelial cells.

The effective delivery of therapeutic proteins to the site of action is of great importance in achieving an effective therapy. Due to hydrophilicity, proteins are generally poorly transported across biological membranes. Chemical acylation represents... expand abstract one of the basic methods for improving their membrane permeability. A novel method for acylation is presented, based on the formation of palmitoylchloride dispersion in aqueous acetonitrile solution, using chicken cystatin as a model protein. We examined the effects of palmitoylchloride/cystatin molar ratio, reaction pH and introduction of successive palmitoylation cycles on the protein modification degree. The reaction products were analysed by capillary electrophoresis and SDS-PAGE, and the in vitro inhibitory activity was determined by N-benzoyl-D,L-arginine-beta-naphthylamide assay. Using cell culture-based assays, we examined the transport properties of unmodified and palmitoylated cystatin, its efficiency to inhibit intracellular enzymes, and its cytotoxicity. We demonstrated that palmitoylated cystatin rapidly internalized into the cell and caused a complete loss of cathepsin B activity. In contrast, the unmodified control cystatin was unable to inhibit the intracellular enzymes. These results strongly suggest protein palmitoylation to be a very effective strategy for improving cell internalization. collapse abstract

Determination of doxycycline in pharmaceuticals and human urine by micellar electrokinetic capillary chromatography.

Micellar electrokinetic capillary chromatography (MEKC) was performed at 25 degrees C and 30 kV (under a pressure of 15 mbar), using 30 mM borate buffer containing 60 mM sodium dodecysulfate (SDS) and 5% (v/v) methanol as background electrolyte (pH 9... expand abstract.0) to determine doxycycline. UV detection was at 350 nm. The method was shown to be specific, accurate (recovery was 100.3 +/- 1.0%), linear over the tested range (correlation coefficient 0.9995) and precise (RSD <1.9%). The method was used to determine doxycycline in tablets, capsules and human urine after oral application. collapse abstract

Engineered staphylococcal protein A's IgG-binding domain with cathepsin L inhibitory activity.

Inhibitory peptide of papain-like cysteine proteases, affinity selected from a random disulfide constrained phage-displayed peptide library, was grafted to staphylococcal protein A's B domain. Scaffold protein was additionally modified in order to al... expand abstractlow solvent exposed display of peptide loop. Correct folding of fusion proteins was confirmed by CD-spectroscopy and by the ability to bind the Fc-region of rabbit IgG, a characteristic of parent domain. The recombinant constructs inhibited cathepsin L with inhibitory constants in the low-micromolar range. collapse abstract

Evaluation of antibacterial activity of extracts of five species of wood-colonizing fungi.

Screening new organisms for antibacterial activity and searching for new antibacterial drugs is important due to the constant generation of new antibiotic-resistant strains of pathogenic bacteria. An E. coli broth microdilution test was used to evalu... expand abstractate the results of the Vibrio fischeri bioluminescence test in five of the most antibacterially active species of wood-colonizing fungi. Serpula lacrymans was found to be a potential source of thermostable antibiotic(s) and the Vibrio fischeri bioluminescence test was confirmed to be a useful method for screening for antibacterial activity. collapse abstract

Computer-aided measurement of psoriatic lesion area in a multicenter clinical trial--comparison to physician's estimations.

BACKGROUND: There is a great need for accurate and informative outcome measures in clinical trials. In psoriasis, therapeutic effect is usually assessed by physician's estimation of psoriatic area and other signs. These measures were found to be un-r... expand abstracteproducible. Several automated procedure for more reproducible measurement of psoriatic area were developed, but were not suitable for large-scale trials. OBJECTIVES: In a multicenter clinical trial, we tested a method where the advantage of accurate computerized measurement of the area on the digital photograph was combined with physician's proficiency. METHODS: The patients with psoriasis in four study centers were included in a placebo controlled clinical trial. They were examined and photographed before and after the therapy with calcipotriol ointment or placebo. The psoriatic area was manually outlined on the patient's photographs and the area was automatically measured by a computer. The areas estimated by physician and measured by computer were compared. RESULTS: We found that computer-aided measurement of psoriatic lesion area improved the power of the clinical trial, compared to the standard approach, where physician's estimations of the psoriatic lesion area tend to overestimate. We also found that adapted PASI index, where the psoriatic area was not converted into an area grade, but was maintained as a continuous variable, also improved the power of the clinical trial. CONCLUSIONS: Computer-aided measurement and an adapted PASI can be used as a powerful and reliable measure to evaluate the effects of antipsoriatic treatment. collapse abstract

Serum procalcitonin levels in patients with mild community-acquired pneumonia.

Comparison of bacterial and phage display peptide libraries in search of target-binding motif.

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-thr... expand abstractoughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better. collapse abstract

Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K.

Endogenous cysteine proteases were given much attention lately, as their role in a variety of pathophysiological disorders became evident. Amongst them cathepsins, which are thought to be implicated in mediation of osteoporosis, cancer progression, a... expand abstracttherosclerosis, and many other conditions, are of considerable interest as drug targets. In the presented work, papain was chosen as a model cysteine protease and panning protocol was optimized for selection of papain-binding phage-displayed peptides from a commercially available combinatorial peptide library. Different selection strategies were applied in order to select high-affinity binders. Ultimately, five cyclic peptides (CNWAAGYNCGGGS-NH2, CWSMMGFQCGGGS-NH2, CWEWGGWHCGGSS-OH, CNWTLGGYKCGGGS-NH2 (all cyclized through formation of intramolecular disulphide bond), and GNWTLGGYKGG (cyclized head-to-tail)) were synthesized and tested for inhibitory activity towards papain and human cathepsins L, B, H, and K. The peptides possess inhibitory constants in the low micromolar to mid-nanomolar range and exhibit certain selectivity for different lysosomal cysteine proteases included in this study. collapse abstract

Determination of cichoric acid content in dried press juice of purple coneflower (Echinacea purpurea) with capillary electrophoresis.

Purple coneflower (Echinacea purpurea) is an immunostimulating drug, containing multiple substances. The most important in activity are polysaccharides, caffeic acid derivatives (cichoric acid), alkamides and glycoproteins. It is not clear yet, which... expand abstract substances are responsible for activity. Cichoric acid is an appropriate marker of the quality of E. purpurea containing product, because it has immune stimulatory effects and it is susceptible to degradation. In this work, an improved capillary electrophoresis method for determining cichoric acid in dried press juice from purple coneflower was developed. The optimal conditions were: electrophoretic buffer-75mM borate, pH 8.8; injection 20mbar for 20s; separation at 20kV; detection at 350nm, temperature 35 degrees C. collapse abstract

Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies.

Interference with fat hydrolysis results in the reduced use of ingested lipids. Inhibition of pancreatic lipase reduces the efficiency of fat absorption in the small intestine and thereby initiates modest long-term reduction in body weight. In an att... expand abstractempt to select peptides with affinity for the surface of pancreatic lipase and potential inhibitory activity, a random, cyclic heptapeptide phage-displayed library was used. Five independent selections, differing in elution step, were performed. In three selection protocols, a sequential elution strategy was applied in anticipation of improving the selection of high-affinity clones. Four heptapeptides with the highest affinity, seemingly for pancreatic lipase, were selected, synthesized, and characterized for their capacity to inhibit enzyme function. Although no clear consensus among the sequenced peptides was found, one of the selected peptides inhibited pancreatic lipase with an apparent inhibition constant of 16 muM. collapse abstract

Screening for antibacterial activity in 72 species of wood-colonizing fungi by the Vibrio fisheri bioluminescence method.

Resistance of pathogenic bacteria to antibiotics leads scientists to discover new antibacterial drugs. Ninety samples of wood-colonizing fungi were cultivated on agar plates, and their extracts tested for antibacterial activity using the Vibrio fisch... expand abstracteri bioluminescence test. Two fungi species, Serpula lacrymans and Nectria vilior, were found to be a potential new source of thermostable antibiotics. Vibrio fischeri bioluminescence test was found to be a useful method for antibacterial activity screening from the samples of natural origin. collapse abstract

Aetiology and clinical presentation of mild community-acquired bacterial pneumonia.

A prospective study was initiated to analyse the bacterial aetiology and clinical picture of mild community-acquired pneumonia in Slovenia using the previously described Pneumonia Severity Index. Radiographically confirmed cases of pneumonia in patie... expand abstractnts treated with oral antibiotics in seven study centres were included. An aetiological diagnosis was attempted using culture of blood and sputum, urinary antigen testing for Streptococcus pneumoniae and Legionella pneumophila, and antibody testing for Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila in paired serum samples. One hundred thirteen patients were evaluable for clinical presentation and 109 for aetiological diagnosis. At least one pathogen was detected in 62.4% patients. The most common causative agents were Mycoplasma pneumoniae in 24.8%, Chlamydia pneumoniae in 21.1%, and Streptococcus pneumoniae in 13.8% of patients. Dual infection was detected in 8.3% of patients. Most patients suffered from cough, fatigue, and fever. Patients with atypical aetiology of pneumonia differed from those with typical bacterial pneumonia or pneumonia of unknown aetiology in age, presence of dyspnea, and bronchial breathing on lung auscultation. Patients with pneumococcal, chlamydial, and mycoplasmal infections differed in age, risk class, presence of dyspnea, bronchial breathing, and proteinuria. There was an overlap of other clinical symptoms, underlying conditions, and laboratory and radiographic findings among the groups of patients classified by aetiology. Since patients with mild community-acquired pneumonia exhibit similar clinical characteristics and, moreover, since a substantial proportion of cases are attributable to atypical bacteria, broad-spectrum antibiotic treatment seems to be recommended. collapse abstract

Rutin in buckwheat herbs grown at different UV-B radiation levels: comparison of two UV spectrophotometric and an HPLC method.

Rutin is an antioxidant with many interesting pharmacological effects. It can also be found in buckwheat (Fagopyrum esculentum Moench). UV radiation stimulates the activity of enzymes of the phenylpropanoid pathway and there is some evidence that it ... expand abstractinfluences the rutin content in plants. The aim of the present research was (1) to examine the influence of different levels of UV-B radiation on rutin content and (2) to compare the results obtained by three analytical methods. The plants were grown under three UV-B levels: reduced, ambient and enhanced, simulating 17% ozone depletion. Analyses were performed by HPLC and two spectrophotometric methods. In one, the absorbancies were measured at 420 nm with and without the addition of AlCl(3). In another method the concentration was calculated from absorbancies at 352.5 nm and 366.5 nm according to the Official Methods of Analysis of AOAC International. The highest amounts of rutin were found in flowers, followed by leaves and stems. A comparison of the different treatments revealed that the highest amounts of rutin were in plants grown under ambient radiation, followed by the plants cultivated under enhanced UV-B and then under reduced UV-B radiation. Treatments caused more effect on leaves than on flowers. Leaves developed under ambient light conditions contained 97% more rutin than leaves grown under reduced UV-B radiation. In flowers, the contents differed by 19% only. The results obtained using the three methods showed a good correlation, but the absolute differences were surprisingly high. The AOAC and the AlCl(3) methods gave, on average, 140% and 30% higher results than HPLC, respectively. collapse abstract

Oxidation of lovastatin in the solid state and its stabilization with natural antioxidants.

Oxidative instability of the hydroxy methylglutaryl Co-A reductase inhibitor lovastatin in the solid state and stabilization with natural antioxidants (ascorbic acid, rutin, gallic acid, quercetin and caffeic acid) was investigated. Lovastatin in the... expand abstract solid state and binary mixtures with 10% (w/w), 25% (n/n), 12.5% (n/n) and 6.3% (n/n) of each of the antioxidants were prepared. Oxidation experiments were performed on the scanning calorimeter using dynamic oxygen atmosphere. The amount of non-oxidized lovastatin was determined using HPLC. The results of the experiments have shown that lovastatin is unstable to oxidation under higher temperatures and in the presence of oxygen, and that some antioxidants markedly stabilize the drug. The most significant antioxidative effect was seen with caffeic acid and rutin, followed by gallic acid and quercetin. Ascorbic acid was only moderately effective. The results prove that flavonoids do have significant antioxidative potential. This phenomenon can be used to improve oxidative stability of drugs such as lovastatin which are sensitive to the presence of oxygen. collapse abstract