Prevention of Fusarium mycotoxin contamination by breeding and fungicide application to wheat.

The two main possibilities for decreasing toxin contamination were investigated in this paper. In the breeding section, we found that for resistance evaluation the ratio of Fusarium-damaged kernels is more important as this has a closer correlation with the deoxynivalenol (DON) content than the extent of Fusarium head blight (FHB). A high variability was found among lines from the non-Fusarium programme. A 50% decrease of DON contamination could be achieved by a simple and consequent resistance control. The tests with the variety candidates proved the same; therefore, the resistance screening for variety registration is an effective means to stop the spreading of highly susceptible genotypes. The resistance breeding programme showed an even larger DON decrease related to regular susceptible control varieties. Fungicide treatments were generally only weakly effective. However, it was shown that the testing methodology was poor, and with the optimal coverage spraying as much as 90% reduction of DON on small plot tests can be achieved. A farm-scale technology was also developed where the DON reduction as a mean for 3 years was higher than 70%, more than double the regular praxis data. To stabilize this efficacy, we need the most powerful fungicides, a nearly horizontal spraying of heads (like Turbo FloodJet nozzles) that receive the necessary coverage and so enough active ingredient to protect heads and the spraying technology should be kept rigorously. A combination of resistance and fungicide application can reduce DON contamination levels to below the European Union limit of 1.25 mg kg(-1) for levels which would otherwise be around 8-10 mg kg(-1). We think that this will solve most of the problems.

Molecular characterization of human adenomyosis.

Adenomyosis is a common gynaecological disorder characterized by the abnormal growth of endometrium into the myometrium and myometrial hypertrophy/hyperplasia. Uterine fibroids are benign neoplasms of the myometrium, and they represent a diagnostic pitfall for adenomyosis. In this study, we have used the genome-wide Affymetrix U133 Plus 2.0 microarray platform to compare the gene expression patterns of adenomyosis, uterine fibroids, normal endometrium and myometrium. Unsupervised principal component analysis (PCA) revealed that these four tissue types could be segregated from one another solely based on their gene expression profiles. Analysis of variance (ANOVA), followed by Tukey means separation test, significance analysis of microarrays (SAM) and 2-fold change threshold, identified 7415 probe sets as differentially expressed among the four groups of samples. Supervised cluster analysis based on these probe sets clustered adenomyosis most closely with endometrium and uterine fibroids with myometrium, consistent with the anatomic origin of these two diseases. The Tukey means separation post hoc testing found 2073 probe sets altered between adenomyosis and normal endometrium or myometrium, and 2327 probe sets altered in expression when comparing uterine fibroids with myometrium. Using Ingenuity Pathways Analysis (IPA), we found 9 highly significant functional networks in adenomyosis and 10 in uterine fibroids. Notably, the top network in both cases was associated with functions implicated in cancer and cell death. Finally, we compared the gene expression profiles of adenomyosis and uterine fibroids and identified 471 differentially expressed probe sets that may represent potential biomarkers for the differential diagnosis of these diseases.

Developmental malformations of the eye: the role of PAX6, SOX2 and OTX2.

Eye development initiates as an evagination of the early neural plate, before the closure of the neural tube. Structural malformations of the eye such as anophthalmia and microphthalmia arise very early in development. It is not surprising therefore that three of the genes currently identified to play a significant role in these developmental eye anomalies are also major players in brain development and regionalization. However, as has been emerging for a high proportion of transcriptional regulators studied, these genes have evolved to play multiple roles throughout development, and perhaps even in adult tissue maintenance. This complex spatiotemporal expression pattern requires elaborate regulatory systems which we are beginning to unravel. A major component of these complex regulatory networks is a series of cis-acting elements, highly conserved through evolution, which spread large distances from the coding region of each gene. We describe how cross regulation for PAX6, SOX2 and perhaps OTX2 has now been uncovered, pointing to the mechanisms that can fine-tune the expression of such essential developmental components. These interactions also help us understand why there is significant phenotypic overlap between mutations at these three loci.

New silicon compounds as resistance modifiers against multidrug-resistant cancer cells.

The efficiency of chemotherapy is often decreased by the development of resistance of cancer cells to cytostatic drugs. This phenomenon is in most cases caused by the activity of the various ABC transporters, multidrug-resistance (MDR) gene-encoded p-glycoproteins, that pump anticancer drugs out of the cells. The inhibition of the activities of the MDR proteins MDR1 and MRP was investigated via the administration of two new organosilicon compounds, alis-409 and alis-421. The study was focused on the inhibition of MDR by blocking the ADR1 gene expression and through the inhibition of the pump-function of mdr-p-glycoprotein, in human breast cancer cell lines expressing mrp and prostate cancer cell line (PC-3). Apoptosis induction and the interaction between epirubicin and the silicon-substituted compounds were studied in human MDR-1 gene-transfected mouse lymphoma and its parent cell line, Colo320/MDR-LRP and sensitive subline Colo205, by means of rhodamine 123 accumulation. The activity of MRP1 p-glycoprotein was studied in human breast cancer cell lines such as HTB-26/MRP1 and two MRP-negative breast cancer cell lines, T47D and MCF7, by carboxyfluorescein accumulation, and on a stomach cancer cell line. The activity of MRP in 257P/MDR and its drug-sensitive derivative were studied in human stomach cancer cells by daunorubicin accumulation in a flow cytometer. The two representative organosilicon derivatives, alis-409 and alis-421, showed antiproliferative effects without apoptosis induction. The drug accumulation in the human MDR1 gene-transfected mouse lymphoma cells was increased without down-regulation of the MDR1 gene expression tested by RT-PCR assay. The rhodamine uptake was increased in L5178/MDR1 and Colo320/MDR1-LRP, but not drug-sensitive human breast cancer MCF-7 and T47D, and L5178 mouse lymphoma parent cells in the presence of alis-409 and alis-421. The MRP-mediated carboxyfluorescein accumulation in HTB-26/MRP human breast cancer cells and daunorubicin accumulation in human stomach cancer cells 257P/MDR were not modified by these alis compounds. A synergistic interaction between epirubicin and the silicon-substituted resistance modifiers was found only in MDR1-mediated MDR in the case of colo-320/MDR1-LRP cells and mouse lymphoma cells transfected with the human MDR1 gene. The results indicate that the organosilyl derivatives specifically act on MDR1 p-glycoprotein 170. The alis compounds act on pgp170 in a way which is similar to verapamil isomers.

Toxoplasma infection and cell free extract of the parasites are able to reverse multidrug resistance of mouse lymphoma and human gastric cancer cells in vitro.

A large number of compounds are known to reduce the ATP-dependent efflux pump activity of multidrug resistant (mdr) tumor cells. Here we report that an infection of cancer cells with T. gondii reduced the multidrug resistance of the tumour cells against cytostatic drugs. Two mouse lymphoma cell lines (Mdr L 5718 and Par 5718) were infected with Toxoplasma gondii in vitro and the reduction of efflux pump activity of the cells was measured. The drug accumulation (Rhodamin-123) was increased in the infected mdr cell lines compared with non- infected mdr-cells, and no effect was shown after infection of the parental cell line. The same effect was also achieved by incubation of Mdr-tumor cells with cell lysate of Toxoplasma gondii. Mdr-1-gene expression was reduced in the infected cell lines 48 hours after infection. Co-cultivation of Toxoplasma gondii with mdr cell lines separated by a microfilter from tumor cells was performed, but this cocultivation did not change the mdr efflux activity. The effect of Toxoplasma gondii infection on the efflux pump activity and mdr-1 gene expression was also examined in the human gastric cancer cells. A sensitization of resistant gastric cancer cells was also achieved by parasite infection. This phenomenon is an evidence that a reduction of resistance in tumor cells can be achieved by a natural parasite infection. It is as yet unclear whether an active infection or another substance of T. gondii is responsible for this phenomenon.

Modulation of MDR1 and CYP3A expression by dexamethasone: evidence for an inverse regulation in adrenals.

A strong overlap exists between gp170 and CYP3A substrates and inducers. In order to investigate a putative coregulation of MDR and CYPA gene expression, we measured their transcripts in human liver and after dexamethasone treatment in HepG2 cells or in different mouse tissues. In human liver, we observed no correlation between MDR1 and CYP3A4 expression, whereas these genes were coinduced by dexamethasone in HepG2 cells. In mouse liver treated with dexamethasone, mdr1b and Cyp3a were induced (5- and 2-fold, respectively). In adrenals, the main expressing gp170 tissue, Cyp3a, was increased while mdr1b was repressed (-51%). The expression of mdr1b increased in heart, brain, and colon and decreased in lung and kidney but Cyp3a was not detectable. In conclusion, human hepatic CYP3A4 and MDR1 are not corregulated but are coinducible. In vivo murine mdr1b and Cyp3a are coregulated by dexamethasone in liver and inversely regulated in adrenals.

Effect of new thioacridine derivatives on P-gp function and on mdr1 gene expression.

We studied the effect of thioacridine derivatives on the function of P-glycoprotein in MDR mouse T-lymphoma cell line L5178 and in MDR human leukemia cell line K562/ADR by rhodamine 123 uptake assay. The effect of some selected thioacridines was also investigated on the expression of the mdr1 gene. Expression was analysed by RT-PCR. Two compounds: 3-amino-9-thio-(4'-nitrobenzyl)acridinone and 2,7-dimethoxy-9-thio-(2'-diethylaminoethyl) acridinone were able to block the function of the P-gp, and also to decrease significantly mdr1 gene expression. Because these two derivatives exert their positive effects as reversing agents they could be potential candidate anticancer agents for further investigation. The thioacridines, which do not affect P-gp function, do not affect or increase the expression of mdr1 gene. Our results showed the structure-activity relationships of these compounds, providing a direction for the development of new, more active compounds.

Tomato lectin labels the 180 kD glycoform of P-glycoprotein in rat brain capillary endothelia and mdr tumor cells.

Two main isoforms of P-glycoproteins can be distinguished according to their solubility in ionic and non-ionic detergents. Studies on mdr cell lines and brain capillary vessels support the evidence that tomato lectin reveals high affinity binding to the oligosaccharide chains of the SDS soluble isoform of P-glycoprotein, but not to the non-ionic detergent soluble isoform. Thus the SDS-soluble isoform represents a glycoform having polylactosamines in its oligosaccharide chains. The function of these oligosaccharides is still unknown, although the carbohydrate chains of P-glycoprotein were believed to take part in correct protein folding only. We also demonstrated that lectin binding to the extracellular lactosamine sequences of drug efflux pump does not change its efficiency on mdr cell lines, but interferes with the inhibitory action of some drugs, such as verapamil and promethazine. In accordance with earlier findings we assume that carbohydrate chains might be involved in stabilization of the active conformation of efflux pump. The possible role of lectin treatment in maintaining P-glycoprotein mediated blood-brain barrier functions has to be proved in further investigations.

The inhibition of SOS-responses and MDR by phenothiazine-metal complexes.

The gene of multidrug resistance (mdr) is inducible by different environmental stresses (SOS gene). We tested the inhibitory action of some new metal complexes of phenothiazines on megacin encoding bacterial gene induced by mitomycin-C as an example of "SOS induction" and on efflux pump of mouse lymphoma cells. The interaction of compounds to DNA was measured by thermal stability of DNA. It was found that metal co-ordination complexes of trifluoperazine (TFP) and chlorpromazine (CPZ) added before mitomycin administration have an inhibitory action on megacine induction. The TFP-V(IV) complex was effective at a lower concentration than TFP alone. The inhibitory effect of some metal coordinating complexes (TFP-Cu(II) and TFP- V(IV)) exceeded the action of TFP alone on efflux pumps. We propose that these compounds can form a complex with the regulatory protein or DNA resulting in the inhibition of SOS response and inhibit the mdr function by inactivating the P-glycoprotein as well.

Drug resistance reversal, anti-mutagenicity and antiretroviral effect of phthalimido- and chloroethyl-phenothiazines.

The effect of substituted phenothiazines was studied in three different systems; bacteria and cancer cells and reverse transcriptase enzyme of Moloney leukemia virus. F'lac and hemolysin plasmids were eliminated by some substituted phenothiazines from E. coli at a very low frequency. The same phenothiazine derivatives also were synergistic with tetracycline in bacteria and shown antimutagenic effect in Ames test. No mutagenic effects were observed in TA 98 strain of Salmonella typhimunium. Chloroethyl-substituted phenothiazines showed antimutagenicity equivalent to the parent compounds; however, phthalimido-substituted phenothiazines had higher antimutagenicity of 50%. P-glycoprotein responsible for multidrug resistance was also inhibited in tumor cells. The accumulation of the fluorescent rhodamine 123 in the phenothiazine treated multi-drug resistant tumor cells was measured by flow cytometry. Some of the substituted phenothiazines were effective P-glycoprotein blockers, while some compounds had moderate activity, but others were without effect as compared to 5 microM verapamil. On the basis of computer analysis there are some correlations between the biological activities and the dipole moments, and entropy of the studied molecules. Our results suggest that the inhibition of Hly+ plasmid replication and P-glycoprotein function may depend partly on similar electronic properties of the studied phenothiazine derivatives. The activity of Moloney leukemia virus reverse transcriptase was inhibited by the substituted phenothiazines, however, no basic differences were found in the activities of phthalimido- and chloroethyl substituted phenothiazines.

Inhibition of the transport function of membrane proteins by some substituted phenothiazines in E. coli and multidrug resistant tumor cells.

Efflux-pumps mediated by P-glycoprotein increase the level of resistance to antibiotics in bacteria and to cytostatics in tumor cells due to decreased drug accumulation, and are also involved in the operation of blood brain barrier. Different compounds are able to enhance drug retention in the cells by inhibiting the efflux-pump mechanism of multidrug resistant (mdr) cancer cells and bacteria. The effects of substituted chlorpromazines were studied on a hemolysin producing and antibiotic resistant plasmid carrying E coli, and rhodamine uptake of multidrug resistant (mdr 1 gene expressing) mouse lymphoma cells. Hemolysin transporter protein encoding plasmids were eliminated from E. coli by a representative phenothiazine namely promethazine. Minimal inhibitory concentrations of tetracyclin and promethazine were lower for plasmidless bacteria as compared to the parent, plasmid carrying strains. The antibiotic resistance plasmid was cured of the R-plasmid of E. coli JE 2571, however, the ring substituted derivatives were less effective then parent compounds. The effect of some substituted phenothiazines on P-glycoprotein efflux-pump of mouse lymphoma cells were studied. The majority of ring substituted derivatives reversed the mdr of tumor cells. The 3,7,8-trihydroxy- and 7,8-dihydroxy derivatives of chlorpromazine were effective as P-glycoprotein blockers, however, 7,8-diacetoxy-, 7,8dimetoxy-, 7-semicarbazone-, and 5-oxo-chlorpromazine derivatives had only moderate effect. A tomato lectin, specific for blood brain capillary endothelium was able to modify the activity of P-glycoprotein in tumor cells. Phenothiazine and tomato lectin had some antagonism in tumor cells. Our results suggest that the inhibition of P-glycoprotein function in murine tumor cells and inhibition of transporter protein in E. coli bacteria may depend on pi-electron superdelocalizibility and electrophile binding of the compounds to the transporter proteins. The intracellular accumulation of antibiotics or chemotherapeutics increased as a consequence of decreased drug efflux in both bacterial and tumor cell systems. The inhibition of the drug effux-pump is the same for all individual cells of the population. These results can be realized by combination chemotherapy, however, antiplasmid effect itself cannot be exploited in this respect because the resistance was reversed in a part of the population only. The similarity with mdr P-glycoprotein in tumor cells and brain capillary endothels provides a good model for molecules opening the blood brain barrier.

Effects of acridines on bacterial plasmid replication and endotoxin.

Different amino- and imino-acridines were systematically synthesized. The antibacterial, antiplasmid, antimotility and endotoxin complexing effect of acridines were studied, when antibacterial effect of the compounds was compared on E. coli. Aminoderivatives were more active than imino-acridines. The N-heptyl-9-imino-acridine was able to select lon minus mutants in the E. coli culture, however, the other acridines tested were ineffective in this respect. The iminoacridines inhibited the motility of Proteus vulgaris more effectively than aminoderivatives. The antimotility action of the acridines was also dependent on the ionic content of the media. The antiplasmid effect was measured on an F-prime plasmid of E. coli LE140 strain. Iminoacridines had a more powerful antiplasmid effect than the amino-substituted derivatives. The majority of the compounds inhibited the intercellular plasmid transfer from E. coli. Kmr donor to a Na-azide resistant recipient. In this test the aminosubstituted derivatives were shown to be more effective inhibitors of conjugation than the imino-substituted compounds. Endotoxin formed complexes with N-butylamino. N-propy-lamino and imidoderivatives. However, complex formation of N-ethyl-, N-heptyl-, N-diethylaminoethyl- and N-diethylamino-propyl-acridines were different. Correlations between molecular orbitals and the antibacterial effects are also discussed.

Synthesis and antitumor activity of 1-[2-(chloroethyl)-3-(2-substituted-10H-phenothiazin-10-yl)alkyl- 1-urea s as potent anticancer agents.

10-[N-(Phthalimido)alkyl]-2-substituted-10H-phenothiazines and 1-(2-chloroethyl)-3-(2-substituted-10H-phenothiazin-10-yl)alkyl-1- ureas were synthesized and found to have antiproliferative effects on human HEp-2 and L5178Y cell cultures. The multi-drug resistant subline of mouse lymphoma was sensitive to the reversal effects of some 10-[N-(phthalimido)alkyl]-2-substituted-10H-phenothiazines, while 1-(2-chloro-ethyl)-3-(2-substituted-10H-phenothiazin-10-yl)alkyl-1 -ureas were less effective but had a similar degree of antiproliferative effect on both cell lines.

Reversal of multidrug resistance by amitriptyline in vitro.

Amitriptyline, a tricyclic antidepressant, was able to reverse the multidrug resistance efflux pump of human colon cancer subline SW 620 and multidrug resistant (mdr) mouse lymphoma cells by decreasing rhodamine 123 efflux. The inhibitory effect of amitriptyline on the efflux pump was dose dependent. An investigation was made of the effects of mouse tumour necrosis factor (TNF) alpha and interferon (IFN) gamma on the efflux pump activity of mdr cells together with amitriptyline compared to the par cells (mdr-). After long-term cytokine pretreatment of mdr cells, the amitriptyline was more effective, due to some synergism between the amitriptyline and TNF-alpha.

Effects of two benzo[a]phenothiazines on multi-drug resistance (mdr) and tumor antigen expression.

Two benzo[a]phenothiazines 5H-benzo[a]phenothiazin-5-one (1), and its derivative 6-methyl-5H-benzo[a]phenothiazin-5-one (2) inhibited the proliferation of human and mouse tumor cell lines. The multi-drug resistant (mdr) subline was more sensitive than its parent cell line to 5H-benzo[a]phenothiazin-5-one (1), 6-methyl-5H-benzo[a]phenothiazin-5-one (2) was equally antiproliferative against the three cell lines tested. Rhodamine 123 efflux of mdr cells was more efficiently inhibited by 5H-benzo[a]phenothiazin-5-one (1) than by 6-methyl-5H-benzo[a]phenothiazin-5-one (2). The exposure of adenovirus infected cells to 5H-benzo[a]phenothiazin-5-one (1) resulted in a reduction of tumor-antigen expression, whereas 6-methyl-5H-benzo[a]phenothiazin-5-one (2) enhanced the T-antigen expression.