Development and In Vivo Application of a Water-Soluble Anticancer Copper Ionophore System Using a Temperature-Sensitive Liposome Formulation.

Liposomes containing copper and the copper ionophore neocuproine were prepared and characterized for in vitro and in vivo anticancer activity. Thermosensitive PEGylated liposomes were prepared with different molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) in the presence of copper(II) ions. Optimal, temperature dependent drug release was obtained at 70:30 DPPC to HSPC weight ratio. Neocuproine (applied at 0.2 mol to 1 mol phospholipid) was encapsulated through a pH gradient while using unbuffered solution at pH 4.5 inside the liposomes, and 100 mM HEPES buffer pH 7.8 outside the liposomes. Copper ions were present in excess, yielding 0.5 mM copper-(neocuproine)2 complex and 0.5 mM free copper. Pre-heating to 45 °C increased the toxicity of the heat-sensitive liposomes in short-term in vitro experiments, whereas at 72 h all investigated liposomes exhibited similar in vitro toxicity to the copper(II)-neocuproine complex (1:1 ratio). Thermosensitive liposomes were found to be more effective in reducing tumor growth in BALB/c mice engrafted with C26 cancer cells, regardless of the mild hyperthermic treatment. Copper uptake of the tumor was verified by PET/CT imaging following treatment with [64Cu]Cu-neocuproine liposomes. Taken together, our results demonstrate the feasibility of targeting a copper nanotoxin that was encapsulated in thermosensitive liposomes containing an excess of copper.

[Oncogen dependent regulation of the migration and proliferation of human tumor cells]. / Humán daganatsejtek migrációjának és proliferációjának onkogénfüggõ szabályozása.

The high mortality of solid tumors can be attributed to their invasive and metastatic potential that is based on their migration and proliferation. Importantly, growth factor receptor (GF) signaling pathways regulating proliferation and migration are often affected by oncogenic mutations and are important targets for antitumor therapy. We found positive correlation between migration and proliferation in melanoma and lung cancer cells using videomicroscopy, not supporting the "go or grow" hypothesis. Furthermore, the invasion into collagen I matrices from brain tumor spheroids was not impaired upon the inhibition of proliferation. Sensitivity of human melanoma cells towards EGF and FGF2 treatment but not against GF receptor tyrosine kinase inhibitors was oncogenic BRAF or NRAS mutation status dependent. Prenylation inhibition failed to decrease clonogenic growth in BRAF mutant but PTEN wild-type melanoma lines but increased migration in BRAF-mutant cells. In certain mesothelioma cells, activin signaling showed a pro-tumorigenic effect suggesting activin as a valuable candidate for therapeutic interference. In summary, our findings demonstrate that proliferation is neither an obstacle nor a prerequisite for tumor cell invasion. Furthermore, the specific oncogenic mutations may differentially regulate migration and proliferation of tumor cells. Therefore, they are not only therapeutic targets but can also profoundly influence the efficacy of various therapies.

Do audio-visual motion cues promote segregation of auditory streams?

An audio-visual experiment using moving sound sources was designed to investigate whether the analysis of auditory scenes is modulated by synchronous presentation of visual information. Listeners were presented with an alternating sequence of two pure tones delivered by two separate sound sources. In different conditions, the two sound sources were either stationary or moving on random trajectories around the listener. Both the sounds and the movement trajectories were derived from recordings in which two humans were moving with loudspeakers attached to their heads. Visualized movement trajectories modeled by a computer animation were presented together with the sounds. In the main experiment, behavioral reports on sound organization were collected from young healthy volunteers. The proportion and stability of the different sound organizations were compared between the conditions in which the visualized trajectories matched the movement of the sound sources and when the two were independent of each other. The results corroborate earlier findings that separation of sound sources in space promotes segregation. However, no additional effect of auditory movement per se on the perceptual organization of sounds was obtained. Surprisingly, the presentation of movement-congruent visual cues did not strengthen the effects of spatial separation on segregating auditory streams. Our findings are consistent with the view that bistability in the auditory modality can occur independently from other modalities.

The effects of rhythm and melody on auditory stream segregation.

While many studies have assessed the efficacy of similarity-based cues for auditory stream segregation, much less is known about whether and how the larger-scale structure of sound sequences support stream formation and the choice of sound organization. Two experiments investigated the effects of musical melody and rhythm on the segregation of two interleaved tone sequences. The two sets of tones fully overlapped in pitch range but differed from each other in interaural time and intensity. Unbeknownst to the listener, separately, each of the interleaved sequences was created from the notes of a different song. In different experimental conditions, the notes and/or their timing could either follow those of the songs or they could be scrambled or, in case of timing, set to be isochronous. Listeners were asked to continuously report whether they heard a single coherent sequence (integrated) or two concurrent streams (segregated). Although temporal overlap between tones from the two streams proved to be the strongest cue for stream segregation, significant effects of tonality and familiarity with the songs were also observed. These results suggest that the regular temporal patterns are utilized as cues in auditory stream segregation and that long-term memory is involved in this process.

Stable individual characteristics in the perception of multiple embedded patterns in multistable auditory stimuli.

The ability of the auditory system to parse complex scenes into component objects in order to extract information from the environment is very robust, yet the processing principles underlying this ability are still not well understood. This study was designed to investigate the proposal that the auditory system constructs multiple interpretations of the acoustic scene in parallel, based on the finding that when listening to a long repetitive sequence listeners report switching between different perceptual organizations. Using the "ABA-" auditory streaming paradigm we trained listeners until they could reliably recognize all possible embedded patterns of length four which could in principle be extracted from the sequence, and in a series of test sessions investigated their spontaneous reports of those patterns. With the training allowing them to identify and mark a wider variety of possible patterns, participants spontaneously reported many more patterns than the ones traditionally assumed (Integrated vs. Segregated). Despite receiving consistent training and despite the apparent randomness of perceptual switching, we found individual switching patterns were idiosyncratic; i.e., the perceptual switching patterns of each participant were more similar to their own switching patterns in different sessions than to those of other participants. These individual differences were found to be preserved even between test sessions held a year after the initial experiment. Our results support the idea that the auditory system attempts to extract an exhaustive set of embedded patterns which can be used to generate expectations of future events and which by competing for dominance give rise to (changing) perceptual awareness, with the characteristics of pattern discovery and perceptual competition having a strong idiosyncratic component. Perceptual multistability thus provides a means for characterizing both general mechanisms and individual differences in human perception.

Predictive regularity representations in violation detection and auditory stream segregation: from conceptual to computational models.

Predictive accounts of perception have received increasing attention in the past 20 years. Detecting violations of auditory regularities, as reflected by the Mismatch Negativity (MMN) auditory event-related potential, is amongst the phenomena seamlessly fitting this approach. Largely based on the MMN literature, we propose a psychological conceptual framework called the Auditory Event Representation System (AERS), which is based on the assumption that auditory regularity violation detection and the formation of auditory perceptual objects are based on the same predictive regularity representations. Based on this notion, a computational model of auditory stream segregation, called CHAINS, has been developed. In CHAINS, the auditory sensory event representation of each incoming sound is considered for being the continuation of likely combinations of the preceding sounds in the sequence, thus providing alternative interpretations of the auditory input. Detecting repeating patterns allows predicting upcoming sound events, thus providing a test and potential support for the corresponding interpretation. Alternative interpretations continuously compete for perceptual dominance. In this paper, we briefly describe AERS and deduce some general constraints from this conceptual model. We then go on to illustrate how these constraints are computationally specified in CHAINS.

Modelling the emergence and dynamics of perceptual organisation in auditory streaming.

Many sound sources can only be recognised from the pattern of sounds they emit, and not from the individual sound events that make up their emission sequences. Auditory scene analysis addresses the difficult task of interpreting the sound world in terms of an unknown number of discrete sound sources (causes) with possibly overlapping signals, and therefore of associating each event with the appropriate source. There are potentially many different ways in which incoming events can be assigned to different causes, which means that the auditory system has to choose between them. This problem has been studied for many years using the auditory streaming paradigm, and recently it has become apparent that instead of making one fixed perceptual decision, given sufficient time, auditory perception switches back and forth between the alternatives-a phenomenon known as perceptual bi- or multi-stability. We propose a new model of auditory scene analysis at the core of which is a process that seeks to discover predictable patterns in the ongoing sound sequence. Representations of predictable fragments are created on the fly, and are maintained, strengthened or weakened on the basis of their predictive success, and conflict with other representations. Auditory perceptual organisation emerges spontaneously from the nature of the competition between these representations. We present detailed comparisons between the model simulations and data from an auditory streaming experiment, and show that the model accounts for many important findings, including: the emergence of, and switching between, alternative organisations; the influence of stimulus parameters on perceptual dominance, switching rate and perceptual phase durations; and the build-up of auditory streaming. The principal contribution of the model is to show that a two-stage process of pattern discovery and competition between incompatible patterns can account for both the contents (perceptual organisations) and the dynamics of human perception in auditory streaming.

Multistability in auditory stream segregation: a predictive coding view.

Auditory stream segregation involves linking temporally separate acoustic events into one or more coherent sequences. For any non-trivial sequence of sounds, many alternative descriptions can be formed, only one or very few of which emerge in awareness at any time. Evidence from studies showing bi-/multistability in auditory streaming suggest that some, perhaps many of the alternative descriptions are represented in the brain in parallel and that they continuously vie for conscious perception. Here, based on a predictive coding view, we consider the nature of these sound representations and how they compete with each other. Predictive processing helps to maintain perceptual stability by signalling the continuation of previously established patterns as well as the emergence of new sound sources. It also provides a measure of how well each of the competing representations describes the current acoustic scene. This account of auditory stream segregation has been tested on perceptual data obtained in the auditory streaming paradigm.

Pharmacognostic research on Viola tricolor L. (Violaceae).

UNLABELLED: In this study we analyzed the chemical composition and we evaluated the diuretic activity of aerial parts of V. tricolor L. MATERIAL AND METHODS: For saponins and mucilages we have performed gravimetric methods, whereas for carotenoids, spectrophotometric determinations and HPLC analysis were performed. We studied the diuretic activity of tincture, using furosemide as the reference drug in male Wistar-Bratislava rats. RESULTS: We established the content in saponins (5.98%), mucilages (14.20%) and total carotenoids (18.46 mg/100 g). In fresh vegetal drug we have determined by HPLC 8 carotenoids: violaxanthin (352 microg/100 g), antheraxanthin (711 microg/100 g), lutein (1575 microg/100 g), zeaxanthin (1488 microg/100 g), alpha-cryptoxanthin (66 microg/100 g), beta-carotene 5,6-epoxide (133 microg/100 g), beta-carotene (1678 microg/100 g) and 9Z-beta-carotene (312 microg/100 g). We observed a moderate diuretic effect of tincture (diuretic index 1.103, saluretic index of Na+ 1.181, and saluretic index of K+ 1.365). CONCLUSIONS: Violae tricoloris herba contains a high amount of saponins and mucilages and it can be recommended to complete the diuretic treatment.

Evaluation of anti-inflammatory activity of alcoholic extract from Viola tricolor.

AIM: Evaluation of anti-inflammatory actions of Viola tricolor aerial parts tincture. MATERIAL AND METHOD: The anti-inflammatory activity of the tincture from Viola tricolor aerial parts was tested in acute inflammation induced with oil of turpentine (i.m. 0.6 ml/100 g b.w.) in male Wistar rats. The results were compared with those from a positive control group with experimental inflammation and with those of a group treated with diclofenac (30 mg/100 g b.w.). We tested the effects by measuring total leukocyte count and differential leukocyte count, a test of in vitro phagocytosis and by the evaluation of nitric oxide (NO) synthesis measuring the metabolites (nitrites and nitrates). RESULTS: Viola tricolor extract (50 mg tincture/100 g b.w.) significantly reduced polymorphonuclear leukocytes and monocytes percentages and the activation of circulating phagocytes. Nitric oxide synthesis had a slight decrease. CONCLUSION: Viola tricolor extract had anti-inflammatory effect on bone marrow acute phase response.

A comparative study of some Veronica species.

UNLABELLED: We have tried to establish some criteria to avoid the substitution of Veronica officinalis (common speedwell) with other species of Veronica genus, especially Veronica chamaedrys (germander speedwell) which is widely spread and has no therapeutic action. MATERIALS AND METHODS: We have studied the differential histological, anatomical and phytochemical characters of these two species. A rapid method for the identification of the two species is the TLC for flavonoids and phenyl-propanic compounds. We have done also a HPLC study, which has permitted the detection of acteoside in Veronica chamaedrys and isoacteoside in Veronica officinalis. In order to confirm the supposed hypocholesterolaemic effect of Veronica officinalis (used in ethno-pharmacy as a hypocholesterolaemic agent) we have done a two step experiment for these two Veronica species. RESULTS: Veronica officinalis in the diet showed no significant effect on the levels of cholesterol and triglycerides in the serum of the cholesterol free diet animals. Veronica officinalis had also a lowering effect on triglycerides and cholesterol level in the serum of high cholesterol diet animals. CONCLUSIONS: A rapid method based on morpho-anatomical and chemical features has been developed in order to avoid substitution of Veronica officinalis with Veronica chamaedrys. For Veronica officinalis we have proved a hypocholesterolaemic effect in high cholesterol diet animals.

The hepatoprotective action of ten herbal extracts in CCl4 intoxicated liver.

The effect of ten phytotherapeutic products on CCl(4) intoxicated liver in albino male Wistar rats was investigated. Biochemical parameters, including serum transaminase activity (GPT and GOT), histoenzymological measurements (lactate dehydrogenase, LDH; succinate dehydrogenase, SDH, cytochromoxidase, CyOx; Mg(2+)-dependent adenosine triphosphatase, ATP-ase) and histochemical (Sudan black) and histological examinations (haematoxylin-eosin staining) of the liver were investigated. Some positive effects such as the reduction of hepatocytolysis and steatosis, and a return to normal values of the activity of some enzymes in the following plants: Chrysanthemum balsamita, Echinacea pallida, Calendula officinalis and Corylus avelana were obtained.

Fast determination of colchicine by TLC-densitometry from pharmaceuticals and vegetal extracts.

Colchicine, (S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo-(a(-heptalen-7-yl)-acetamide, is the main alkaloid contained in Colchicum autumnale (meadow saffron). There are known colorimetric, spectrophotometric, volumetric, potentiometric, voltametric, gravimetric and various chromatographic methods for quantitative determination of colchicine, each of them presenting a series of advantages and disadvantages. As an alternative, we proposed the use of a densitometric determination for colchicine allowing the determination of this alkaloid from pharmaceutical products, as well from seeds of meadow saffron. The total alkaloid extract was separated by Thin-Layer Chromatography using Silicagel 60F(254) layers and a mixture of chloroform:acetone:diethylamine (5:4:1) as mobile phase. The same conditions were used for the determination from pharmaceutical products. Densitometric measurements were carried out at the absorption maximum (350 nm) of colchicine, the determinations being made by reflectance and by fluorescence. The peaks were optimized regarding to their area and shape by varying four scanning parameters (slit width and height, number of measurements and scanning speed). We established the calibration plot using pure colchicine in the range 50-600 ng mL(-1). The proposed method could be widely used in the pharmaceutical industry for the quick and accurate quantitative determination of colchicine because it eliminates the interferences given by other bioactive or degradation compounds. The method was characterized by validation parameters (linearity, accuracy, fidelity, sensitivity) and it was established its performances in comparison with an HPLC method and an official quantitative determination from the Romanian Pharmacopoeia X edition respectively.

Root, stem and leaf anatomy of Geranium palustre Torner Cent. (Geraniaceae).

An anatomical study on the root, stem and leaf of Geranium palustre Torner Cent. has been performed in order to continue our previous researches on this species. Technique of inclusion into paraffin and optical microscope have been used. Root, stem and leaf transverse sections were studied. They contain calcium oxalate crystals as inclusions. The root presents elements of secondary structure (suber, secondary cortex, secondary xylem and phloem). The vascular bundles in the stem can be classified as internal or external and differ in the presence or absence of an own sclerenchyma sheet depending on their age. The vascular bundles of stem are collateral. Leaf structure is bi-facial. Stem and leaf surface have no glandular hairs.

Preliminary HPLC study on some polyphenols of Geranium robertianum L. (Geraniaceae).

In order to continue our previous studies concerning Geranium robertianum L., herb Robert (Geraniaceae), we have realised a HPLC study of some polyphenols using an original method created by a group of young researchers from the University of Medicine and Pharmacy of Cluj-Napoca. We have identified and measured in the dried Geranii robertiani herba (harvested from Valea Runcului, district of Alba-Iulia) the following compounds: hyperoside (3.64 microg/100 mg), ellagic acid (7599.76 microg/100 mg), isoquercitrin (49.49 microg/100 mg), quercetrin (83.92 microg/100 mg), kaempferols (143.43 microg/100 mg), caftaric acid (166.92 microg/100 mg), rutoside (72.23 microg/100 mg). We have also analysed a hydrolysed sample of the same drug in which we have identified and measured: caffeic acid (6.62 microg/100 mg), ellagic acid (10550.65 microg/100 mg), quercetrin (203.44 microg/100 mg), kaempferols (231.80 microg/100 mg), caftaric acid (47.41 microg/100 mg). We have indirectly proved the presence of ellagic tannins (the amount of ellagic acid increases after acid hydrolise) and the existence of bi- or polycaffeoil derivatives (the caffeic acid is present only in the hydrolysed sample). The flavonoid aglycones exist in both forms: as free compounds and as part of the flavonoid molecules.

Mechanisms involved in metalloid transport and tolerance acquisition.

Toxic metalloids such as arsenic and antimony have always been an integral part of the natural environment. To survive in such a hostile habitat, it is crucial to develop strategies to exclude toxic substances from the cell and to acquire tolerance. Cells remove metalloids from the cytosol either by active efflux or by sequestration in an internal organelle. Controlling the influx appears to be another way of maintaining a low intracellular metalloid content. Inside the cell, the metalloid can be reduced to a form that is recognised by the expulsion system(s). In addition, metalloid complexation and compartmentalisation contributes to enhanced cellular tolerance. Finally, the presence of metalloids activates transcription of various cellular defence genes. Metalloid-containing drugs are currently used to treat protozoan infections and promyelocytic leukaemia. Since metalloid resistance hampers efficient treatment, interest in identifying the mechanisms involved in tolerance acquisition has arisen. The possibility of using genetic approaches has made the yeast Saccharomyces cerevisiae a compelling model system to investigate the basis of metalloid tolerance at a molecular level. This review describes the recent progress made in elucidating the mechanisms involved in metalloid transport and tolerance in yeast and other organisms.

The glycerol channel Fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae FPS1 gene encodes a glycerol channel protein involved in osmoregulation. We present evidence that Fps1p mediates influx of the trivalent metalloids arsenite and antimonite in yeast. Deletion of FPS1 improves tolerance to arsenite and potassium antimonyl tartrate. Under high osmolarity conditions, when the Fps1p channel is closed, wild-type cells show the same degree of As(III) and Sb(III) tolerance as the fps1Delta mutant. Additional deletion of FPS1 in mutants defective in arsenite and antimonite detoxification partially suppresses their hypersensitivity to metalloid salts. Cells expressing a constitutively open form of the Fps1p channel are highly sensitive to both arsenite and antimonite. We also show by direct transport assays that arsenite uptake is mediated by Fps1p. Yeast cells appear to control the Fps1p-mediated pathway of metalloid uptake, as expression of the FPS1 gene is repressed upon As(III) and Sb(III) addition. To our knowledge, this is the first report describing a eukaryotic uptake mechanism for arsenite and antimonite and its involvement in metalloid tolerance.

The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage.

A major part of the transcriptional response of yeast cells to osmotic shock is controlled by the HOG pathway and several downstream transcription factors. Sko1p is a repressor that mediates HOG pathway-dependent regulation by binding to CRE sites in target promoters. Here, we report five target genes of Hog1p-Sko1p: GRE2, AHP1, SFA1, GLR1 and YML131w. The two CREs in the GRE2 promoter function as activating sequences and, hence, bind (an) activator protein(s). However, the two other yeast CRE-binding proteins, Aca1p and Aca2p, are not involved in regulation of the GRE2 promoter under osmotic stress. In the absence of the co-repressor complex Tup1p-Ssn6p/Cyc8p, which is recruited by Sko1p, stimulation by osmotic stress is still observed. These data indicate that Sko1p is not only required for repression, but also involved in induction upon osmotic shock. All five Sko1p targets encode oxidoreductases with demonstrated or predicted roles in repair of oxidative damage. Altered basal expression levels of these genes in hog1Delta and sko1Delta mutants may explain the oxidative stress phenotypes of these mutants. All five Sko1p target genes are induced by oxidative stress, and induction involves Yap1p. Although Sko1p and Yap1p appear to mediate osmotic and oxidative stress responses independently, Sko1p may affect Yap1p promoter access or activity. The five Sko1p target genes described here are suitable models for studying the interplay between osmotic and oxidative responses at the molecular and physiological levels.

Essential oils analysis. II. Mass spectra identification of terpene and phenylpropane derivatives.

Mass spectra are widely used in order to identify the peaks resulting from a chromatographic separation. The most common approach to solve the problem for unknowns on whom very little other structural information is available is the use of a retrieval algorithm and a reference mass spectra database. The wide variety of mass spectra recorded with different instruments under various experimental conditions can lead to erroneous results. In order to improve the accuracy of the results, we proposed earlier an identification algorithm, which combines the information obtained from both GC and MS fingerprints. This paper presents a new algorithm based on the comparison of the unknown mass spectra with several libraries (including Wiley and NIST) by using reverse and direct search algorithms respectively. The results of the comparisons were quantified with respect to the match quality and the interference compounds. A global match index for the comparison using all the above information was computed and the results were presented as the match probability. This index expresses more accurately the matches between unknown and all the available libraries mass spectra. In order to verify our algorithm, we tried to identify the compounds separated by GC-MSD from different species of Acorus calamus L. (Araceae) essential oils. The probability of the matches increases compared with the quality of matches resulting from Wiley and NIST libraries.

Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans.

In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3-phosphate dehydrogenase (G3PDH)-encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3-phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin-interacting agent calcofluor and the membrane-destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi.