Synthesis and biological evaluation of 8-hydroxy-2,7-naphthyridin-2-ium salts as novel inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).

By analogy with the natural product chelerythrine, which has been identified as an inhibitor of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), we prepared a small series of 8-hydroxy-2,7-naphthyridin-2-ium salts. Spectroscopic analyses allowed us to elucidate the zwitterionic nature of 2,7-naphthyridin-1(7H)-ones, the neutral state of 8-hydroxy-2,7-naphthyridin-2-ium salts. Among the tested compounds, we identified dual inhibitors of AChE and BChE as well as an inhibitor showing a preferential inhibition of AChE over BChE. By in vitro characterization in combination with docking studies, we were able to identify structural features that influence the biological activity of 8-hydroxy-2,7-naphthyridin-2-ium salts.

First trimester hyperglycosylated human chorionic gonadotrophin in serum - a marker of early-onset preeclampsia.

INTRODUCTION: Recent studies indicate that treatment with low-dose aspirin may reduce the risk of preeclampsia. Thus, early prediction of preeclampsia is needed. Low serum concentrations of hyperglycosylated human chorionic gonadotrophin (hCG-h) are associated with early pregnancy loss. We therefore studied whether it may serve as an early marker of preeclampsia. METHODS: A nested case-control study included 158 women with subsequent preeclampsia, 41 with gestational hypertension, 81 normotensive women giving birth to small-for-gestational-age (SGA) infants and 427 controls participating in first trimester screening for Down's syndrome between 8 and 13 weeks of gestation. Gestational-age-adjusted multiples of medians (MoMs) were calculated for serum concentrations of hCG-h, the free beta subunit of hCG (hCGß) and pregnancy-associated plasma placental protein A (PAPP-A) and the proportion of hCG-h to hCG (%hCG-h). Clinical risk factors including mean arterial pressure (MAP) and parity were also included in the risk calculation. RESULTS: In women with subsequent preeclampsia %hCG-h was lower than in controls (median MoM 0.92, P < 0.001), especially in 29 cases with early-onset preeclampsia (0.86, P < 0.001), in which PAPP-A also was reduced (0.95, P = 0.001). At 90% specificity for prediction of early-onset preeclampsia, sensitivity was 56% (95% confidence interval, 52-61%) for %hCG-h, 33% (28-37%) for PAPP-A, and 69% (51-83%) for the combination of these with first trimester MAP and parity. The area under the receiver-operating characteristic (ROC) curve for the combination of all these was 0.863 (0.791-0.935). CONCLUSIONS: hCG-h is a promising first trimester marker for early-onset preeclampsia. Addition of PAPP-A and maternal risk factors may improve the results.

OS009. Second trimester serum hyperglycosylated human chorionic gonadotrophin and preeclampsia.

INTRODUCTION: Hyperglycosylated human chorionic gonadotrophin (hCG-h), produced by the placental trophoblast cells, is involved in placental development in early pregnancy. Decreased second trimester urine hCG-h is associated with later preeclampsia, which may be a sign of impaired trophoblastic invasion preceding symptoms of the disease. OBJECTIVES: To study whether maternal second trimester serum hCG-h concentrations predict later development of preeclampsia. METHODS: Fifty-five women with subsequent preeclampsia, 21 women with gestational hypertension, 30 normotensive women with small-for-gestational-age (SGA) infants and 83 controls with uneventful pregnancies were included in the study. Their serum hCG and hCG-h concentrations were analyzed by fluoroimmunoassay at 14-17weeks of gestation. The proportion of hCG-h of total hCG (%hCG-h) was calculated and converted to multiples of the median (MoMs) of the controls. MoMs of the groups were compared by Mann-Whitney U test. Pearson's correlation was used to analyze correlations between clinical characteristics and serum marker concentrations. The results are given as medians with 95% confidence intervals (95% CI). A two-tailed P<0.05 was considered significant. RESULTS: The concentrations of hCG-h and %hCG-h decreased with advancing gestational weeks in women with subsequent preeclampsia (r=-0.289, p=0.032 and r=-0.464, p<0.001), but not in women in the other groups. There was a tendency towards lower concentrations of hCG-h and %hCG-h in women with subsequent preeclampsia than in controls. The median MoMs of %hCG-h were 0.89 (95% CI,0.79-1.00) in women with subsequent preeclampsia and 1.00 (0.91-1.11) in controls. The corresponding values for women with subsequent gestational hypertension were 1.00 (0.86-1.16), for those with subsequent SGA infants they were 1.09 (0.89-1.23). The difference between preeclampsia and the other groups together was significant (p=0.029). CONCLUSION: Earlier studies suggest that decreased urine hCG-h concentrations reflect changes in placental function that precede the development of preeclampsia. At 14-17weeks of gestation, the serum concentrations of hCG-h showed moderate validity to predict later development of preeclampsia. Further studies on the utility of hCG-h for prediction of subsequent preeclampsia are warranted.

Changes in serum concentrations of soluble vascular endothelial growth factor receptor-1 after pregnancy.

BACKGROUND: Vascular endothelial growth factor (VEGF) is the most potent stimulator of angiogenesis. It mediates its activity through two membrane-bound receptors, VEGFR-1 and VEGFR-2, both expressed in the placenta. Beginning in early pregnancy, the soluble form of the first, sVEGFR-1, binds and inhibits most of the biological actions of circulating VEGF. After delivery, it disappears from the maternal circulation. METHODS: We determined the biological elimination rate of endogenous sVEGFR-1 after term pregnancy in serial venous samples obtained during and after elective Caesarean sections (n=8), and we demonstrated the relationship between serum sVEGFR-1 and VEGF after mid-trimester legal termination of pregnancy (n = 5), by analysing their concentrations using immunoassays (ELISA). RESULTS: The disappearance of sVEGFR-1 from circulation after Caesarean delivery was biphasic with a rapid half-life of 3.4 h (2.2-7.5 h; median, range) and a slow one of 29 h (17-94 h). After mid-trimester legal termination of pregnancy the sVEGFR-1 concentrations decreased and those of free VEGF simultaneously increased with a highly significant negative correlation with each other (r = -0.90, P < 0.0001). CONCLUSIONS: The disappearance of endogenous sVEGFR-1 after pregnancy is biphasic, and it is associated with a simultaneous increase in free VEGF concentrations.

Development of an automated 7-day 96-well Caco-2 cell culture model.

Caco-2 cells are a widely accepted model to predict permeability and absorption of compounds in humans. We built up an automated 96-wellplate Caco-2 permeation model with reduced growth time. Model compounds (metoprolol, ketoprofen, verapamil, naproxen, hydrochlorthiazide), permeability markers (TEER, Lucifer Yellow, D-[1-(14)C]-mannitol) and confocal microscopy were used to assess the utility of our method on Biomek-FX-automation. The confocal imaging data showed that Caco-2 cells formed monolayers when cultured for 7 days with initial cell density of 2.1 x 10(5) cells/cm2. P-Glycoprotein was present in Caco-2 cells, localized on the plasma membrane. Permeation of model compounds was comparable to those obtained from traditional 12-wellplate experiments.

Preliminary antiproliferative effects of some species of Terminalia, Combretum and Pteleopsis collected in Tanzania on some human cancer cell lines.

Methanolic extracts (25 microug/ml) of species belonging to the genera of Combretum, Terminalia and Pteleopsis, collected during a field expedition in Tanzania in 1999, were screened for their antiproliferative and cytotoxic effects against three human cancer cell lines (HeLa, cervical carcinoma; T 24, bladder carcinoma; and MCF 7, breast carcinoma). A leaf extract of Combretum fragrans and a fruit extract of C. zeyheri gave the strongest antiproliferative and cytotoxic effects of all the twenty-four extracts screened in this investigation. In contrast to the highly powerful leaf extract of C. fragrans, the root extract of this species gave no cytotoxic effects against the investigated cancer cell lines at a concentration of 25 microg/ml. The other investigated species of Combretum and Terminalia differed greatly in their cytotoxic potential. Root extracts of Terminalia sambesiaca and T. sericea gave the strongest cytotoxic effects of the five species of Terminalia used in this study. Eight of the twenty-four investigated plant extracts showed pronounced cytotoxic effects (<30% proliferation compared to the control) against the T 24 bladder cancer cells, seven against the HeLa cells and four against the MCF 7 cells.

Identification of COMT and ErmC inhibitors by using a microplate assay in combination with library focusing by virtual screening.

The paper describes a process of facilitated screening by using a combination of molecular modelling and a 96-well microplate assay for the identification of novel inhibitors of catechol-O-methyltransferase (COMT) and bacteria expressing ErmC. With the help of virtual screening the number of compounds processed in the in vitro screening assay was reduced from over 200,000 to 49. Out of the 49, two structurally very similar compounds were identified as confirmed hits with reasonable activity (IC50 values of 26 and 73 microM) and thus as potential core structures for further drug design and development.

Effects of the aqueous extract of Bryothamnion triquetrum on chemical hypoxia and aglycemia-induced damage in GT1-7 mouse hypothalamic immortalized cells.

The neuroprotective ability of the aqueous crude extract of Bryothamnion triquetrum (S. G. Gmelin) Howe and its cinnamic acids was studied in GT1-7 cells exposed to the combination of chemical hypoxia (KCN 3 mM) and aglycemia conditions. These ischemia-like conditions provoked acute and delayed cytotoxicity in GT1-7 cells if extended for more than 90 min. The extract was able to protect from the cell death produced by severe (180 min) chemical hypoxia/aglycemia insult, which cannot be related to its glucose content, and also reduced the cytotoxicity and early production of free radicals produced by mild (105 min) insult. Results showed that some of these protective effects of the extract are partially related to the presence of ferulic acid. The data additionally suggest that neuroprotection exerted by the extract is related to its ability to reduce free-radical generation by mechanisms different from the direct scavenging of the radical entities.

Inhibitory effect of dietary phenolic compounds on Chlamydia pneumoniae in cell cultures.

Chlamydial infections are very common worldwide. All chlamydial species have a tendency to cause persistent infections, which have been associated to several chronic diseases including blinding trachoma, infertility and coronary heart disease (CHD). At present, no efficient treatment for the eradication of chronic chlamydial infections exists and, thus, new antichlamydial compounds are urgently needed. This study was designed to screen antichlamydial activity of natural flavonoids and other natural and structurally similar synthetic compounds against Chlamydia pneumoniae in human cell line (HL). HL cells were infected with C. pneumoniae and incubated 72 h with studied compounds. Reduction in the number of inclusions was determined with immunofluorescence staining. In vitro minimum inhibitory concentration was also determined for some of the most active compounds. Thirty seven percentage of the studied compounds (57 in total) were highly active against C. pneumoniae and all the studied compounds were non-toxic to the host cells at studied concentrations. Our study revealed direct antichlamydial effect for selected polyphenolic compounds against C. pneumoniae, in vitro. We also demonstrated the ability of some of the investigated compounds to accumulate inside cells or into cell membranes and cause inhibition, even when present only prior to infection.

A rapid screening method for detecting active compounds against erythromycin-resistant bacterial strains of Finnish origin.

A rapid and simple microdilution technique on 96-well microplate based on turbidimetry was optimized and validated for screening of antimicrobial activity against erythromycin-resistant bacterial strains of Streptococcus pyogenes and Staphylococcus simulans isolated from Finnish patients. Using S. pyogenes ATCC 12351 as reference strain the developed method was evaluated by reproducibility measurements and using parameters typically employed for screening methods, i.e. signal-to-background, signal-to-noise and a screening-window coefficient, the Z' factor. The method was further used for screening a group of natural compounds and their synthetic derivatives against resistant bacterial strains. Of these, octyl and dodecyl gallates, and usnic and ursolic acids were the most active. The described method is a rapid, homogeneous, cost-effective and easy-to-perform system for screening of new potential antimicrobial agents in drug discovery.

Tubifex tubifex Müll.--photosensitive organism.

The worm Tubifex tubifex Müll. (Tubificideae, Oligochaeta) is a suitable organism for the research of the biological effect of various pollutants. This pilot study deals with the responds of the organism to the treatments of two photosensitizers (bengal rose B, quinidine) and UVA radiation. The activity of the photosensitizers was evaluated by the comparison of the surfaces of tested worms and dark controls. The results showed that T. tubifex Müll. could be a suitable organism for the studies of phototoxicity. This species demonstrated relatively strong sensitivity to the effect of the selected photodynamically active substances.

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus.

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania.

An ethnobotanical investigation on the medicinal uses of some species of Terminalia and Combretum (Combretaceae) was carried out in Mbeya, Tanzania during a 5-weeks field expedition. Of the sixteen species collected, Combretum fragrans F. Hoffm., Combretum molle G. Don., Combretum psidioides Welw., Combretum zeyheri Sond., Terminalia kaiserana F. Hoffm. and Terminalia sericea Burch ex. DC. have medical applications against various bacterial infections, such as gonorrhoea and syphilis, and against symptoms like diarrhoea, hypertension and even cancer. Antimicrobial screening of the crude extracts of the selected Combretum and Terminalia species was performed by the agar diffusion method. Among the most effective extracts were methanol extracts of the roots of Terminalia sambesiaca Engl. & Diels., T. kaiserana Guill. & Perrott., T. sericea Burch. ex DC., C. fragrans F. Hoffm. and Combretum padoides Engl. & Diels., all of which showed marked inhibition against Gram-positive bacteria, and were also good inhibitors of Enterobacter aerogenes. All four of the extracts of the roots of T. sericea tested, (methanol, ethanol, acetone and hot water) had good antimicrobial activity. A methanolic leaf extract of T. kaiserana was the only extract to have a bacteriocidic effect on Escherichia coli. Methanol root extracts of T. sambesiaca and methanol leaf extracts of T. sericea were the most effective against Candida albicans. The results of the antimicrobial screening support the ethnomedical uses of these plants.

Isolation and biological activity of frankiamide.

An antibiotic produced by the symbiotic actinomycete Frankia strain AiPs1 was isolated from culture broth using optimized thin-layer chromatography and high-performance liquid chromatography (HPLC) methods. The novel compound that was isolated, dubbed frankiamide, displayed antimicrobial activity against all 14 Gram-positive bacterial strains and six pathogenic fungal strains tested. The pathogenic actinomycete Clavibacter michiganensis and the oomycete Phytophthora were especially susceptible. In addition to displaying antimicrobial activity, frankiamide also strongly inhibited 45Ca(2+) fluxes in clonal rat pituitary GH4C1 tumor cells and was comparable to a frequently used calcium antagonist, verapamil hydrochloride. The results of HPLC analysis, supported by both nuclear magnetic resonance and mass spectroscopy studies, showed that frankiamide has a high affinity for Na(+) ions.

The coumarin osthol attenuates the binding of thyrotropin-releasing hormone in rat pituitary GH4C1 cells.

The influence of two plant coumarins, osthol and xanthotoxin, on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH were studied in clonal rat pituitary GH4C1 cells. Osthol, but not xanthotoxin, decreased the TRH-induced transient increase in [Ca2+]i in Fluo-3 loaded cells incubated in Ca(2+)-free buffer. Binding experiments with [3H]TRH showed that osthol decreased the binding of TRH to its receptor, whereas the affinity of the receptor for TRH increased. This resulted in a decreased TRH-evoked production of IP3 in cells treated with osthol, and a decreased mobilization of sequestered calcium. Osthol did not inhibit the release of calcium evoked by exogenous IP3 in permeabilized cells. Furthermore, osthol decreased the uptake of 45Ca2+ in response to high K+. Xanthotoxin had no effects in these experiments. The results show that osthol modulates TRH-evoked responses by interacting with the TRH receptor.

Effects of simple aromatic compounds and flavonoids on Ca2+ fluxes in rat pituitary GH(4)C(1) cells.

The biological activity of phenolic compounds from plants is well documented in vitro, but little is known about the possible effect of simple aromatic compounds and flavonoids on voltage-operated Ca2+ channels (VOCCs). In pituitary cells, several intracellular pathways may regulate the activity of VOCCs. In this study, we investigated the effect of nine phenylpropanes and metanes, and 20 flavonoids on high K(+)-induced 45Ca2+ entry in clonal rat pituitary GH(4)C(1) cells. At the highest dose tested (20 microg/ml), flavone (a flavone) inhibited 45Ca2+ entry by 63.5%, naringenin (a flavanone) by 56.3% and genistein (an isoflavone) by 54.6%. The phenylmetane derivative octyl gallate was the most potent compound tested, with an IC(50) value of 15.0 microg/ml. The IC(50) value for the reference compound verapamil hydrochloride was 3.0 microg/ml. In sharp contrast to the above, the flavonols quercetin and morin potentiated 45Ca2+ entry. At 20 microg/ml, quercetin increased 45Ca2+ entry by 54.1% and morin by 48.0%. Quercetin increased the cellular cAMP content in a concentration-dependent manner. H 89, an inhibitor of protein kinase A, inhibited the effect of quercetin on 45Ca2+ entry. The results thus suggest that the effect of quercetin is the result of a protein kinase A-mediated activation of VOCCs. Quercetin induced a rapid and marked increase in both the transient (143.1+/-4.2%) and delayed (198.8+/-10.0%) Ca2+ currents, measured by the whole cell patch clamp technique. The onset of the inhibitory effect of octyl gallate was slow, but resulted in an almost complete inhibition of both Ca2+ currents.

Expression of vascular endothelial growth factor receptors 1, 2 and 3 in placentas from normal and complicated pregnancies.

Extensive angiogenesis and invasion of the maternal decidua by trophoblasts are essential for the development and function of the placenta. Vascular endothelial growth factors (VEGF), placenta growth factor (PlGF) and their receptors VEGFR-1/Flt-1, VEGFR-2/KDR and VEGFR-3/Flt4 have important roles in vasculogenesis and angiogenesis. We have studied the localization of these proteins by immunohistochemistry and Western blotting in the placenta and of PlGF in maternal serum, and their association with diabetes, pre-eclampsia, fetal growth restriction (FGR) and fetal alcohol syndrome (FAS). VEGFR-1 and VEGFR-3 were detected mainly in the syncytiotrophoblastic layer whereas VEGFR-2 was detected in the vascular endothelial cells of the placenta. VEGFR-1, but not the other receptors, showed increased expression in placental syncytiotrophoblasts from 50% of patients with severe pre-eclampsia and FGR when compared with normal placentas. PlGF was undetectable in 38 of 44 samples of amniotic fluid of mothers with normal and complicated pregnancies. However, maternal serum PlGF concentrations were significantly lower in pre-eclamptic patients and in those with FGR when compared to diabetic women or healthy controls. These results suggest that low maternal serum PlGF and increased placental expression of its receptor VEGFR-1 are associated with pre-eclampsia and FGR.

Effect of an in vitro fertilization program on serum CA 125, tumor-associated trypsin inhibitor, free beta-subunit of human chorionic gonadotropin, and common alpha-subunit of glycoprotein hormones.

OBJECTIVE: To investigate the impact of an IVF program on serum levels of tumor markers CA 125, tumor-associated trypsin inhibitor, free hCG beta-subunit, and free glycoprotein hormone alpha-subunit. DESIGN: A prospective controlled clinical study. SETTING: Outpatient university infertility clinic. PATIENT(S): Seventy-one infertile patients (with tubal occlusion, pelvic endometriosis, or unexplained infertility) undergoing IVF and nine control women with regular menstrual cycles. INTERVENTION(S): Serial blood sampling before, during, and after IVF, or during one ovulatory menstrual cycle in the controls. MAIN OUTCOME MEASURE(S): Serum levels of CA 125, tumor-associated trypsin inhibitor, hCG-beta, and glycoprotein hormone-alpha. RESULT(S): Before IVF, all tumor markers were within the normal range except for CA 125, which was elevated in patients with endometriosis. IVF led to significant increases in CA 125 and glycoprotein hormone-alpha that differed from the changes seen during normal menstrual cycles. The luteal phase increase in CA 125 correlated with levels of E(2) and P and the number of follicles. Two months after IVF, levels of CA 125 were 12% higher than levels before treatment. Tumor-associated trypsin inhibitor and hCG-beta revealed no cyclicity. CONCLUSION(S): An IVF regimen increased the release of CA 125 and glycoprotein hormone-alpha. The CA 125 elevation after IVF implies a persistent effect of ovarian hyperstimulation on CA 125 release.