Interactions of norharman and harman with DNA.

The interactions of norharman (9H-pyrido [3,4-b] indole) and harman (1-methyl-9H-pyrido [3,4-b] indole) with DNA were studied. DNA caused remarkable fluorescence quenching and change in the absorption spectra of the dyes. Scatchard plots obtained by optical titration gave Kd values of 2.2 X 10(-5)M and 7.7 X 10(-6)M, and apparent numbers of binding sites of 0.13/base and 0.12/base for norharman and harman, respectively. Agarose gel electrophoresis of circular DNA, closed in the presence or absence of norharman revealed that the dye intercalates DNA, thereby causing 17 +/- 3 degrees unwinding of the double helix.     

Exposure to beta-carbolines norharman and harman

The aromatic beta-carbolines norharman and harman have been implicated in a number of human diseases including Parkinson's disease, tremor, addiction and cancer. It has been shown that these compounds are normal body constituents formed endogenously but external sources have been identified. Here, we summarise literature data on levels of norharman and harman in fried meat and fish, meat extracts, alcoholic drinks, and coffee brews. Other sources include edible and medicinal plants but tobacco smoke has been identified as a major source. Exposure levels from these different dietary sources are estimated to a maximum of 4 microg norharman per kg body weight (bw) per day and 1 microg harman per kg bw per day. Exposure via tobacco smoke depends on smoking habits and type of cigarettes but can be estimated to 1.1 microg/kg bw for norharman and 0.6 microg/kg bw for harman per package of cigarettes smoked. Studies on toxicokinetics indicate that inhalative exposure leads to a rapid increase in plasma levels and high bioavailability of norharman and harman. Oral bioavailability is lower but there are indications that sublingual absorption may increase dietary uptake of beta-carbolines. Endogenous formation can be estimated to be 50-100 ng/kg bw per day for norharman and about 20 ng/kg bw per day for harman but these rates may increase with high intake of precursors. Biomarker studies on plasma levels of beta-carbolines reported on elevated levels of norharman, harman or both in diseased patients, alcoholics and following tobacco smoking or consumption of beta-carboline-containing food. Cigarette smoking has been identified as major influence but dietary exposure may contribute to exposure.     

Effects of harman and norharman on the metabolism and genotoxicity of 2-acetylaminofluorene in cultured rat hepatocytes

Monolayers of rat hepatocytes metabolize 0.25 m M 2-acetylaminofluorene (AAF) to various ether-extractable, water-soluble as well as covalently bound products. The major ether-extractable metabolite formed is 2-aminofuorene (AF), followed by 7-OH-AAF and 9-OH-AAF. Pretreatment of rats with the inducer Aroclor 1254 (PCB) increased the metabolism of AAF and caused an increased DNA repair synthesis in hepatocytes exposed to AAF or AF. With N-OH-AAF, a decreased genotoxic response in PCB-treated cells compared to control cells was seen. The addition of harman and norharman decreased the metabolism of AAF to ether-extractable metabolites, water-soluble metabolites and metabolites covalently bound to macromolecules. In contrast, the DNA-repair synthesis caused by the same concentrations of AAF was increased by harman. One explanation for this apparent discrepancy could be that the aromatic amines changed the metabolism of harman and norharman in such a way that these compounds were converted into genotoxic metabolites.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-ydroxy-2-acetylaminofluorene - PCB polychlorinated biphenyls, Aroclor 1254 - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin - TdR thymidine - Trp-P-1 3-amino-1,4dimethyl-5H-pyrido(4,3b)indole - Trp-P-2 3-amino-l-methyl-5H-pyrido(4,3b)indole - UDS unscheduled DNA synthesis     

Effects of harman and harmine on naloxone-precipitated withdrawal syndrome in morphine-dependent rats

The effects of the beta-carbolines, harman and harmine, on naloxone-precipitated withdrawal syndrome in morphine-dependent rats were investigated. Two morphine pellets containing 75 mg morphine base were implanted subcutaneously in the scapular area of adult male Wistar rats (200-250 g) under light ether anesthesia. Rats were then assigned to several groups (n = 12 for each group). Seventy-two hours after morphine implantation, harman (5 and 10 mg/kg), harmine (5 and 10 mg/kg) or saline was injected to rats intraperitoneally (ip). After 45 min, a morphine withdrawal syndrome was precipitated by naloxone (2 mg/kg, ip), and morphine withdrawal signs were observed and evaluated for 15 min. Harmine (5 and 10 mg/kg) attenuated significantly the intensity of all signs of morphine withdrawal except for jumping. While jumping behaviour appearing in morphine withdrawal was intensified by harman (5 and 10 mg/kg) treatment, harmine administration did not produce any significant change in the intensity of this sign. Harman attenuated significantly the intensity of wet dog shakes, writhing, defecation, tremor and ptosis. However, it produced no significant changes in the intensity of teeth chattering and diarrhea. Our results suggest that harman and harmine, beta-carbolines, have some beneficial effects on naloxone-precipitated morphine withdrawal syndrome in rats. Findings from the present study also indicated that harmine was more effective than harman on morphine abstinence syndrome.     

Enhancing effects of heterocyclic amines and beta-carbolines on the induction of chromosome aberrations in cultured mammalian cells.

The effects of post-treatment with heterocyclic amines and beta-carbolines on the induction of chromosome aberrations were studied in Chinese hamster CHO K-1 cells and SV40-transformed excision repair-deficient human XP2OSSV cells. The number of chromosome aberrations induced by UV and MMC were increased by post-treatment with Trp-P-1 and Trp-P-2, in both the presence and the absence of S9 mix. A alpha C, MeA alpha C, Glu-P-1, Glu-P-2, IQ, MeIQ, harman and harmine increased chromosome aberrations only in the presence of S9 mix. Glu-P-2, IQ, MeIQ, harman, and harmine did not induce chromosome aberrations by themselves at the concentrations used in this study. Trp-P-1, Trp-P-2, A alpha C, MeA alpha C and Glu-P-1 were weak clastogens by themselves, but at much higher concentrations than those at which they increased the induction of chromosome aberrations in cells pretreated with UV or MMC. Therefore, the increases in chromosome aberrations were not considered to be additive.     

Identification of the sex pheromone of Isoceras sibirica Alpheraky (Lepidoptera, Cossidae)

We discovered that extracts of the female sex pheromone gland of the carpenterworm moth Isoceras sibirica Alpheraky, a pest of Asparagus officinalis Linn., contained (Z)-7-tetradecen-1-ol (Z7-14:OH), (Z)-9-tetradecen-1-ol (Z9-14:OH), (Z)-7-tetradecenyl acetate (Z7-14:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), and (Z)-9-hexadecadecenyl acetate (Z9-16:Ac). The average levels of the chemicals in a single sex pheromone gland of a calling moth were (0.71 +/- 0.24) ng, (1.42 +/- 0.44) ng, (4.36 +/- 0.32) ng, (8.71 +/- 0.26) ng, and (0.82 +/- 0.38) ng, respectively. The electroantennography (EAG) analysis of these chemicals and their analogues demonstrated that Z9-14:Ac triggered significantly the male EAG response. Traps with rubber septa lure impregnated with Z9-14:Ac (500 microg/septum), Z7-14:Ac (250 microg/septum), and Z9-16:Ac (50 microg/septum) were more effective in catching male moths than traps with other baits or virgin females. Addition of Z7-14:OH and Z9-14:OH to rubber septa did not enhance the efficiency of the trap.     

[3H]Harman Binding Experiments. II: Regional and Subcellular Distribution of Specific [3H]Harman Binding and Monoamine Oxidase Subtypes A and B Activity in Marmoset and Rat

[3H]Harman (1-[3H]methyl-beta-carboline) was used in a novel radioligand binding assay to label selectively and with high affinity monoamine oxidase (MAO) type A. The concentration of the enzyme was determined in six CNS regions of the primate species marmoset (Callithrix jacchus) and of the rat: hypothalamus, hippocampus, cerebellum, cerebral cortex, striatum, and spinal cord. The specific [3H]harman binding in the CNS of the marmoset reveals the same pharmacological profile and other characteristics (affinity, saturability, and reversibility) as in the CNS of the rat. The regional distribution of the [3H]harman binding density (Bmax) in the CNS exhibits a distinct pattern in the marmoset and the rat and a 35 (hypothalamus) to 75% (hippocampus) lower Bmax in the marmoset than in the rat. The Bmax values of [3H]harman binding in the CNS of the marmoset and the rat combined as well as those from visceral organs of the rat (liver, heart, lung, thymus, spleen, and kidney) correlated positively and highly significantly with the respective Vmax values of specific MAO activity of the A type but not of the B type, determined with kynuramine as the substrate. In subcellular fractionation experiments with rat cerebral cortex, the highest [3H]harman binding density (Bmax) and MAO-A activity (Vmax) were detected in mitochondrial fractions and severalfold lower values in the synaptosomal membrane fraction. In conclusion, we suggest that [3H]harman binding is a biochemical tool as a selective marker to quantify MAO-A in the CNS of different mammalian species as well as in extraneuronal tissues.     

Red blood cells: reservoirs of cis- and trans-epoxyeicosatrienoic acids

Epoxyeicosatrienoic acids (EETs) are candidate endothelium-derived hyperpolarizing factors that demonstrate a wide range of biological effects. The presence of both cis- and trans-EETs in rat plasma was identified with HPLC-electrospray ionization tandem mass spectrometry in this study. The total EETs in plasma are 38.2 ng/ml with cis-EETs representing 21.4 +/- 0.4 ng/ml and trans-EETs 16.8 +/- 0.4 ng/ml. EETs in RBCs were estimated to be 20.2 ng/10(9) RBCs, which corresponds to 200 ng in RBCs contained in 1 ml blood. RBC incubation with 10 mM tert-butyl hydroperoxide resulted in 4.4-fold increase of total cis-EETs (from 9.2 to 40.2 ng/10(9) RBCs) and 5.5-fold increase of total trans-EETs (from 11.0 to 60.8 ng/10(9) RBCs). EETs were released (2 ng/ml) from RBCs after incubation at 37 degrees C for 10 min even after being washed 3 times, indicating that RBCs are reservoirs of plasma EETs. The identification of cis- and trans-EETs in RBCs and in plasma as well as their release from RBCs suggest a vasoregulatory role of RBCs in view of their potent vasoactivity.     

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.     

Comparison of the response of midgut epithelial cells and cell lines from lepidopteran larvae to CryIA toxins from Bacillus thuringiensis

The cytotoxic responses of midgut epithelial cells (MEC) from spruce budworm (SBW), gypsy moth (GM) and silkworm (SW) larvae were compared with the cytotoxic response of lepidopteran cell lines (SF-9, SE-1a, and CF-1) to CryIA toxins from Bacillus thuringiensis. The MEC from SBW, SW and GM had binding proteins for CryIA(a,b,c) toxins, whereas the lepidopteran cell lines had binding proteins for CryIA(c). Single MEC exposed to CryIA(a,b,c) toxins in a qualitative lawn assay were equally susceptible to the toxins with a threshold response at about 1ng. The cell lines were not susceptible to CryIA(a,b) toxins in the dose range tested, but had threshold responses for CryIA(c) of 3.4ng for SF-9, 50.2ng for SE-1a and 5.9ng for CF-1. In the quantitative Live/Dead assay, MEC were equally susceptible to CryIA(a,b,c) toxins with a threshold effect at about 1ng and a maximum effect at about 10ng. CF-1 was most sensitive to CryIA(c) with a threshold effect at 0.39ng and a maximal effect at about 1ng. In contrast, a 25-50 times greater dose of CryIA(a) or CryIA(b) was required to elicit a similar response as CryIA(c) for CF-1. SF-9 and SE-1a were most susceptible to CryIA(c) with a threshold effect observed at about 0.5ng and maximal effects at about 2ng. SF-9 cells have a threshold and maximum response to CryIA(a,b) of about 10ng and 20ng, respectively. SE-1a cells have a threshold and maximal response to CryIA(a,b) of 5ng and 10ng, respectively. Intact midgut epithelium exposed to CryIA(a,b,c) toxins had a threshold dose of 2ng for CryIA(b), 10-30ng for CryIA(a) and 2-30ng for CryIA(c). This study has shown that MEC are affected by a broader spectrum of toxins compared to the lepidopteran larvae and insect cell lines.     

催吐萝芙木中生物碱的研究

从催吐萝芙木根的乙醇提取物中分离得到15个吲哚生物碱,利用波谱(ESI-MS,1H NMR,13C NMR)等技术分别鉴定为利血平(1),四氢鸭脚木碱(2),异山德维辛碱(3),利血平酸甲酯(4),萝芙木碱(5),山德维辛碱(6),异育亨宾(7),霹雳萝芙木碱(8),α-育亨宾(9),育亨宾(10),催吐萝芙木定(11),四叶萝芙新碱(12),harman(13),mauiensine(14),12-hydroxymauiensine(15)。其中化合物13 ~15是首次从该植物中分离到。     

[3H]Harman Binding Experiments. I: A Reversible and Selective Radioligand for Monoamine Oxidase Subtype A in the CNS of the Rat

Harman (1-methyl-beta-carboline) is an endogenous compound with neurotropic properties in rats and humans. In a novel in vitro binding assay, the binding site of [3H]harman has been characterized in the rat crude mitochondrial (P2) fraction. The binding was saturable and reversible. Only a single high-affinity binding site was detected by kinetic, saturation, and displacement analyses in the cerebral cortex of the rat. The linear Scatchard plots revealed equilibrium dissociation constant (KD) values of approximately 2.5 nM at 0 degrees C, approximately 9 nM at 23 degrees C, and approximately 30 nM at 37 degrees C. Among six CNS regions (hypothalamus, hippocampus, cerebral cortex, striatum, cerebellum, and spinal cord), the highest density of binding sites (Bmax) was determined in the hypothalamus (approximately 5.5 pmol/mg of protein) and the lowest in the spinal cord (approximately 2.0 pmol/mg of protein). Several drugs known to affect serotonergic, adrenergic, dopaminergic, cholinergic, or GABAergic neurotransmission inhibited specific binding at best in the micromolar range. In contrast, potent and selective inhibitors of monoamine oxidase subtype A were active in the lower and middle nanomolar range. The displacing potency (apparent Ki) of substrates and inhibitors of monoamine oxidase correlated positively and highly significantly with the corresponding values of the inhibition of monoamine oxidase activity of subtype A (r = 0.92, p less than 0.001, n = 17) but not of subtype B (r = -0.47, p greater than 0.05, n = 15). In conclusion, [3H]harman was identified as a specific ligand of the active site of the A subtype of monoamine oxidase in rat brain.     

Chlorination of harman and norharman with sodium hypochlorite and co-mutagenicity of the chlorinated products

Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water. It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o-toluidine with S9 mix. When these beta-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced. Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o-toluidine toward Salmonella typhimurium TA98 with S9 mix. These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.     

Cytokine profile in cases with premature elevation of progesterone serum concentrations during ovarian stimulation

The aim of this study was to investigate the concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), leptin, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-6, in cycles with a premature rise of serum progesterone. 25 intracytoplasmic sperm injection (ICSI) cycles with (Group 1) and 25 ICSI cycles without a premature progesterone elevation (Group 2) were included. The cut-off value of serum progesterone on the day of human chorionic gonadotropin (hCG) administration was 0.9 ng/ml. The indication for ICSI was male factor infertility exclusively. On the day of hCG injection, serum IL-6, VEGF and bFGF were significantly higher in Group 1 (7.7+/-24.5 pg/ml, 290.2+/-161.4 pg/ml and 15.7+/-8.2 ng/ml respectively) than in Group 2 (1.7+/-0.7 pg/ml, 175.2+/-92.1 pg/ml, and 9+/-1.6 ng/ml respectively). On the day of follicular puncture, serum cytokine concentrations were similar in the two groups. IL-6 intrafollicular concentrations were higher in Group 1 (14.7+/-20.7 pg/ml) than in Group 2 (9+/-9.3 pg/ml, p=0.031). There were no differences regarding the ICSI outcome. Patients with serum progesterone above 0.9 ng/ml, have elevated serum concentrations of IL-6, VEGF, and bFGF, as well as elevated intrafollicular concentrations of IL-6. The outcome of ICSI cycles is not associated with premature elevation of progesterone when the cut-off value is set at 0.9 ng/ml.     

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) is induced in rabbit articular chondrocytes by cotreatment with interleukin 1 beta and a protein kinase C activator.

The synthesis of an 88-kDa gelatinolytic enzyme, identified as a zymogen of matrix metalloproteinase (proMMP)-9, was induced in the primary culture of rabbit articular chondrocytes by cotreatment with recombinant interleukin 1 beta (rIL-1 beta) and the protein kinase C (PKC) agonists, phorbol 12,13-dibutyrate (PDBu) or mezerein. Negligible 88-kDa gelatinolytic activity was produced by unstimulated cells or cells treated with a PKC activator alone at concentrations up to 100 ng/ml, and only a modest induction occurred with rIL-1 beta alone at concentrations of 1-100 ng/ml. However, when these cells were treated with a PKC activator in the presence of IL-1 beta (1 ng/ml), induction was striking, with enzymic activity detectable at a concentration as low as 1 ng/ml of mezerein or 10 ng/ml of PDBu. Rabbit chondrocytes in culture constitutively produced the zymogen of MMP-2 (proMMP-2) and its production was not altered by treatment with IL-1 beta or PKC agonists alone or in combination. Recombinant tumor necrosis factor alpha (rTNF alpha) did not substitute for IL-1 beta in inducing proMMP-9 in the presence of PKC activators, nor was the combination of IL-1 beta or TNF alpha alone effective. These data indicate that rabbit articular chondrocytes have a potential to synthesize and secrete proMMP-9 under certain biological and pathological conditions but that the expression of proMMP-9 is differently regulated from that of other MMPs.     

Stage-specific expression of bone morphogenetic protein type I and type II receptor genes: Effects of follicle-stimulating hormone on ovine antral follicles

We investigated the mRNA expression patterns of receptor genes for bone morphogenetic proteins-15 (BMP15) and growth differentiation factor-9 (GDF-9) in granulosa cells of sheep treated with FSH. The effects of FSH and estradiol (E2) on the regulation of BMPRII, BMPRIB and ALK-5 in ovine granulosa cells were also examined. Ovaries were collected on day 16 of the estrous cycle and granulose cells were harvested from follicles of two sizes (3-5 and >5mm in diameter). For in vitro studies, granulosa cells were obtained from follicles of 3-5mm in diameter and cultured in serum-free McCoy's 5A medium supplemented with different doses of FSH (0, 1, 5, 10ng/ml) or a combination of 5ng/ml FSH with 1ng/ml E2. Expression of BMPRII, BMPRIB and ALK-5 mRNA was estimated by quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than of small follicles. Treatment of granulose cells with FSH (1-10ng/ml) alone down-regulated the expression of BMPRIB (P<0.05). BMPRII and ALK-5 mRNA expression was not significantly different at an FSH concentration of 5ng/ml compared to control. A further increase in FSH (10ng/ml) down-regulated the expression of BMPRII and ALK-5 (P<0.05). The combination of FSH (5ng/ml) and E2 (1ng/ml) up-regulated the expression of BMPRII, BMPRIB and ALK-5 in granulose cells (P<0.05). Therefore, the present study establishes the expression levels of the receptor genes of BMP15 and GDF-9 and suggests that the expression of BMPRII, BMPRIB and ALK-5 may be regulated by FSH and E2 in ovine granulosa cells.     

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.     

In vivo modulation of platelet deposition on human atherosclerotic lesions by various antiaggregatory prostaglandins

The influence of intravenous infusions of various prostaglandins on in vivo platelet function was studied after labelling of autologous platelets with 100 mu ci 111 indium-oxinesulfate in patients with peripheral vascular disease stage II according to FONTAINE. PGI2 (5 ng/kg/min) provoked a significant decrease of platelet deposition and a prolongation in platelet half-life time (74 +/- 6 vs 68 +/- 5 hours). PGE1 (25 ng/kg/min) failed to influence platelet deposition, but prolonged significantly platelet half-life time (82 +/- 6 vs 76 +/- 8 hours). CG 4203 (25 ng/kg/min) decreased significantly platelet deposition and prolonged significantly platelet half-life time (73 +/- 10 vs 67 +/- 11 hours). Iloprost (1 and 2 ng/kg/min) reduced significantly platelet deposition without dose relation. Half-life time was increased significantly after therapy compared to placebo (1 ng: 76 +/- 7 vs 69 +/- 7; 2 ng: 73 +/- 9 vs 67 +/- 9 hours).     

High-performance liquid chromatographic determination of 9-(3-pyridylmethyl)-9-deazaguanine (BCX-34) in biological fluids

9-(3-Pyridylmethyl)-9-deazaguanine (BCX-34), a new purine nucleoside phosphorylase inhibitor, has selective immunosuppressive activity with potential therapeutic value in T-cell-mediated diseases. We now report a sensitive, specific and reproducible method for measurement of 9-(3-pyridylmethyl)-9-deazagunanine in biological fluids using high-performance liquid chromatography (HPLC). 9-(3-Pyridylmethyl)-9-deazagunanine was extracted from plasma using perchloric acid precipitation followed by passage through Sep-Pak C₁₈ cartridges (average extraction efficiency, 64.6%). Standard curves were linear over the range of interest (28–1120 ng/ml in plasma and 200–4000 ng/ml in urine, r²>0.999). Within-day and between-day coefficients of variation were less than 8%. The limit of quantitation was 28 ng/ml in plasma and 200 ng/ml in urine. This HPLC method should be useful in future clinical studies with this drug.