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.

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.

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.

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.

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.

Unbound vascular endothelial growth factor and its receptors in breast, human milk, and newborn intestine.

BACKGROUND: Human milk, rich in cytokines, may contain the potent permeability- and angiogenesis-promoting agent vascular endothelial growth factor (VEGF). OBJECTIVE: We wanted to study whether free or bound VEGF is present in human milk and whether it and its receptors (VEGFR-1 and -2) are expressed in lactating breast or newborn intestinal tissue. DESIGN: The study had a longitudinal design with collection of human milk from healthy (n = 32) and diabetic (n = 5) women at 2, 7, and 30 d postpartum. Milk was analyzed for VEGF by enzyme-linked immunosorbent assay along with plasma samples collected 2 d postpartum. Immunohistochemistry was used to localize VEGF and its receptors in lactating breast and newborn intestine. Gel filtration with radiolabeled VEGF was performed to study whether human milk contains VEGF binding proteins. RESULTS: Human milk VEGF concentrations in healthy (76 +/- 19 microg/L, x +/- SD) and diabetic (75 +/- 25 microg/L) women did not differ at 2, 7 (23 +/- 7 and 27 +/- 8 microg/L, respectively), or 30 d (14 +/- 5 and 17 +/- 7 microg/L, respectively) postpartum. VEGF was undetectable in all but 3 plasma samples. Human milk was free of VEGF binding proteins. VEGFR-1 and -2 immunoreactivity was seen in the glandular epithelial cells of the newborn intestine and lactating breast, whereas VEGF was present only in breast glandular epithelium. CONCLUSIONS: The high concentrations of VEGF in human milk, especially colostrum, are not affected by maternal diabetes and may play a role in newborn nutrition.

Differential inhibition of coumarin 7-hydroxylase activity in mouse and human liver microsomes.

Coumarin is 7-hydroxylated by the P450 isoform Cyp2a-5 in mice and CYP2A6 in humans. Various drugs, endogenous substances, plant substances and carcinogens, altogether about 90 chemicals, were evaluated as possible inhibitors of coumarin 7-hydroxylase (COH) activity in mouse microsomes. The effects of selected compounds on COH activity in human liver microsomes were also tested. The furanocoumarin derivatives methoxsalen (8-methoxypsoralen) and psoralen proved to be the most potent inhibitors of mouse COH activity (IC50 values 1.0 and 3.1 microM, respectively). The furanocoumarins bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), imperatorin and sphondin also effectively inhibited mouse COH activity (IC50 values 19-40 microM). Methoxsalen, isopimpinellin and metyrapone were also inhibitors in mice in vivo. Methoxsalen was a potent inhibitor of COH activity also in human liver microsomes, (IC50 value 5.4 microM), whereas bergapten, isopimpinellin and imperatorin had no effect. The imidazole antimycotic miconazole was a potent but non-specific inhibitor of COH activity. Several known substrates and inhibitors of members in the CYP1A, CYP2B, CYP2C, CYP2D and CYP3A subfamilies were poor inhibitors of COH activity. These results suggest that (i) the coumarin-type compounds in particular interact with the active sites of Cyp2a-5 and CYP2A6, and (ii) the active sites of Cyp2a-5 and CYP2A6 are structurally different, since a number of compounds inhibited mouse, but not human COH activity.

Amniotic fluid--soluble vascular endothelial growth factor receptor-1 in preeclampsia.

OBJECTIVE: To measure the levels of the soluble receptor for the potent angiogenic agent vascular endothelial growth factor (VEGF) in amniotic fluid (AF) in healthy and complicated pregnancies, and compare them with levels of erythropoietin, another factor upregulated by hypoxia. METHODS: We assessed amniotic fluid from the second (n = 35, gestational weeks 14-19) and third (n = 29) trimesters of healthy women, and from the third trimesters of preeclamptic (n = 22) and diabetic women with (n = 11) or without preeclampsia (n = 34) and from women with fetal growth restriction (FGR) (n = 14) for soluble VEGF receptor-1 (VEGFR-1) by enzyme-linked immunosorbent assay. RESULTS: In early normal pregnancy, AF-soluble VEGFR-1 levels were higher (median 22 ng/mL, range 2.3-29.5 ng/mL) than in the third trimester (median 13 ng/mL, range 0.5-32 ng/mL; P < .05). In preeclamptic women during the third trimester, levels were higher (median 20 ng/mL, range 10.5-37 ng/mL; P < .05) than healthy controls. The lowest third-trimester levels were in diabetic women (median 11 ng/mL, range 0.5-27 ng/mL). In women with preeclampsia and diabetes, AF-soluble VEGFR-1 levels remained lower (median 13, range 6-32 ng/mL; P < .05) than in women with preeclampsia alone. Amniotic fluid levels of soluble VEGFR-1 in women with FGR (median 19.5 ng/mL, range 5-40 ng/mL) did not statistically differ from those of controls. The AF levels of soluble VEGFR-1 did not correlate with those of erythropoietin. Soluble VEGFR-1 was clearly detectable (median 14 ng/mL, range 9-22 ng/mL) in culture media from placental biopsies (n = 20). CONCLUSION: Preeclampsia is associated with increased levels of soluble VEGFR-1, which are independent of erythropoietin, another hypoxia-inducible factor.

Endothelial tie receptor antigen in maternal and cord blood of healthy and preeclamptic subjects.

OBJECTIVE: To measure the vascular endothelial receptor tyrosine kinase Tie, essential in the process of angiogenesis, in blood from healthy and preeclamptic pregnant women, in umbilical cord blood from both, and in blood from nonpregnant women. METHODS: A total of 143 women participated in four arms of the study. Blood samples were collected from 54 healthy nonlaboring pregnant women (gestational weeks 14-41). Samples were collected immediately prepartum and postpartum from another 40 healthy women (15 delivered vaginally and 25 by cesarean) and 15 preeclamptic women (all delivered by cesarean). Arterial and venous cord samples were collected, when possible, from infants born by cesarean. Single blood samples were drawn from 34 nonpregnant controls. Of these, weekly samples from 11 were drawn during one menstrual cycle. A time-resolved fluoroimmunoassay was developed for the detection of the soluble extracellular domain of Tie. RESULTS: Maternal serum Tie levels decreased with advancing gestational age after 26 weeks (r = .6, P < .001). They were significantly higher in healthy women at term (median 233 ng/mL, range 152-414 ng/mL) compared with nonpregnant controls (median 173 ng/mL, range 107-333 ng/mL, P < .001) or with preeclamptic women at term (median 152 ng/mL, range 90-372 ng/mL, P < .05). This difference between healthy and preeclamptic women persisted on the first postpartum day (median 221 ng/mL, range 128-343 and median 152 ng/mL, range 90-372 ng/mL, respectively, P < .05). The highest levels of serum Tie receptor were observed in umbilical arterial and venous blood (median 240 ng/mL, range 174-474 ng/mL and median 340 ng/mL, range 245-690 ng/mL, respectively). In nonpregnant women, serum Tie levels did not vary with menstrual cycle. CONCLUSION: The high levels of the extracellular domain of Tie in healthy term maternal and cord blood may indicate a role for Tie in the vascular development of human fetuses and placentas.

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.

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.

High-performance liquid chromatography methods for the separation and quantitation of spironolactone and its degradation products in aqueous formulations and of its metabolites in rat serum.

HPLC assays have been developed for the determination of spironolactone and its degradation products 7 alpha-thiospirolactone and canrenone in aqueous solutions of beta-cyclodextrins and for the determination of spironolactone and its metabolites 7 alpha-thiospirolactone, 7 alpha-thiomethylspirolactone, 6 beta-hydroxy-7 alpha-thiomethylspirolactone and canrenone in rat serum samples. Both methods were well suited for their respective applications, i.e., studying the stability of spironolactone in liquid formulations of beta-cyclodextrins and the oral absorption of spironolactone in rats. The HPLC method developed for spironolactone and its metabolites in rat serum requires very small volumes of serum making it possible to take several blood samples over a period of time.

Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds.

Plant phenolics, especially dietary flavonoids, are currently of growing interest owing to their supposed functional properties in promoting human health. Antimicrobial screening of 13 phenolic substances and 29 extracts prepared from Finnish plant materials against selected microbes was conducted in this study. The tests were carried out using diffusion methods with four to nine microbial species (Aspergillus niger, Bacillus subtilis, Candida albicans, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Staphylococcus aureus and Staphylococcus epidermidis). Flavone, quercetin and naringenin were effective in inhibiting the growth of the organisms. The most active plant extracts were purple loosestrife (Lythrum salicaria L.) against Candida albicans, meadowsweet (Filipendula ulmaria (L.) Maxim.), willow herb (Epilobium angustifolium L.), cloudberry (Rubus chamaemorus L.) and raspberry (Rubus idaeus L.) against bacteria, and white birch (Betula pubescens Ehrh.), pine (Pinus sylvestris L.) and potato (Solanum tuberosum. L.) against gram-positive Staphylococcus aureus.

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.

Optimization of selectivity in high-performance liquid chromatography using desirability functions and mixture designs according to PRISMA.

A computer program for the mobile phase optimization of high-performance liquid chromatography (HPLC) is described. The desirability function technique combined to the prisma mixture design was employed to enhance the quality of HPLC separations. The use of statistical models to predict the behaviour of retention times (tR) and band broadening at the different eluent compositions obtained by prisma was examined for dansyl amides and coumarins. The study showed that the dependence between the eluent composition and tR values of dansyl amides and coumarins can be expressed using quadratic regression models with a high degree of accuracy. Band broadening given by means of the band width at half-height (wh) was described by a linear regression model. Both models were used in calculating and predicting the resolution (Rs) in various solvent combinations. The desirability function converted the calculated (Rs) value into the desirability value (D), and the overall optimum was then defined by means of the overall desirability. The optimal eluent mixtures for the separation of compounds were easily read from the contour plot inside the horizontal plane of the prisma model. A good separation was achieved using the optimized solvent combination. Depending on the aims of the optimal separation, the program allows either optimization of critical pairs or achieving the overall optimum giving a reasonable separation for as many compounds as possible.

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.

Bioactivity of certain Egyptian Ficus species.

The fruit extracts of Ficus sycomorus L., F. benjamina L., F. bengalensis L. and F. religiosa L. were screened for bioactivity. F. bengalensis and F. religiosa demonstrated activity in the brine shrimp test (Artemia salina) which indicates toxicity, whereas F. sycomorus and F. benjamina showed no activity. All the fruit extracts exhibited antitumor activity in the potato disc bioassay. None of the tested extracts showed any marked inhibition on the uptake of calcium into rat pituitary cells GH4C1. The extracts of the four tested Ficus species had significant antibacterial activity, but no antifungal activity. The results of this preliminary investigation support the traditional use of these plants in folk medicine for respiratory disorders and certain skin diseases.