Possible mechanism of the stimulatory effect of Artemisia leaf extract on the proliferation of cultured endothelial cells: involvement of basic fibroblast growth factor

To investigate the possible mechanism of the stimulatory effect of a hot water extract from Artemisia leaf (Artemisia princeps PANPANINI) (AFE) on the proliferation of endothelial cells, cells from bovine aorta were cultured for 72 h in RPMI1640 medium supplemented with 10% fetal calf serum in the presence of 5 micrograms/ml AFE. The AFE treatment significantly increased the cell number after culture, while in the presence of 10 micrograms/ml unfractionated heparin, AFE conversely decreased it. This implied that AFE enhanced the cell growth promotion by basic fibroblast growth factor (bFGF). The accumulation of bFGF was significantly increased in the culture medium, in the low-affinity (glycosaminoglycans-binding) fraction, and in the cell extract fraction, but was unchanged in the high-affinity (receptor-binding) fraction. The contents of [35S]sulfate-labeled glycosaminoglycans in both cell layer and the medium were not increased by AFE treatment. The proliferation of A10 cells, an established cell line of smooth muscle cells from murine aorta, was not stimulated by AFE. A10 cells did not produce a significant amount of bFGF in the presence or absence of AFE. Thus, the production of bFGF was considered to be involved in AFE stimulation of cell proliferation. In conclusion, it was suggested that AFE stimulated endothelial cell proliferation by increasing the production of bFGF rather than by an increase in the number of bFGF receptors and the content of glycosaminoglycans in the cell layer. The enhanced reserve of bFGF in the low-affinity fraction of cell layer and in the medium would cause the AFE-stimulated proliferation of endothelial cells.     

Gardenia fruit extract does not stimulate the proliferation of cultured vascular smooth muscle cells, A10

We investigated the effect of a hot water extract from Gardenia fruit (Gardenia jasminoides Ellis) (GFE), which has a stimulatory effect on endothelial cell proliferation, on the proliferation of A10 cells, an established cell line of vascular smooth muscle cell from murine aorta in a culture system. GFE did not change the number of A10 cells after a 48 h culture. GFE significantly increased the incorporation of [3H]thymidine and [14C]leucine into the acid-soluble fraction of bovine aortic endothelial cell layers, but significantly decreased that of A10 cells. These results suggested that GFE stimulates the proliferation of endothelial cells but not of A10 cells. In the endothelial cell culture, GFE significantly increased the accumulation of basic fibroblast growth factor, which is an autocrine for endothelial cell proliferation in medium and low-affinity (glycosaminoglycans-binding) fractions, while A10 cells did not produce a significant amount of the factor. Since it is postulated that a selective stimulation of endothelial cell proliferation by increasing the production of basic fibroblast growth factor is appropriate for prevention of arteriosclerosis and thrombosis, GFE may contain a beneficial component as a useful drug.     

Stimulants from gardeniae fructus for cultured endothelial cell proliferation.

Bioassay-guided fractionation of Gardeniae Fructus extract (GFE), which stimulates the proliferation of cultured endothelial cells, led to the isolation of glycerol and D-mannitol. Both compounds significantly increased the incorporation of [3H]thymidine and [14C]leucine into the acid-insoluble fraction of bovine aortic endothelial cell layers in culture. This clearly indicated that glycerol and D-mannitol are active components of GFE on endothelial cell proliferation. On the other hand, they did not change the number of cultured vascular smooth muscle cells from bovine aorta. Glycerol and D-mannitol may be beneficial drugs for vascular disorders.     

High-performance liquid chromatographic analysis of 5-ethylpyrimidines and 5-methylpyrimidines in plasma

A high-performance liquid chromatographic (HPLC) method employing a C18 reversed-phase column, a mobile phase of sodium acetate and methanol, and an ultraviolet detector was developed for the analysis of 5-ethylpyrimidines and 5-methylpyrimidines in plasma. Samples were prepared for HPLC by sequential cation-exchange and anion-exchange column chromatography. Linear standard curves were obtained for samples containing 0.05-50 micrograms/ml 5-ethyl-2'-deoxyuridine and 5-ethyluracil, 0.05-10 micrograms ml 5-(1-hydroxyethyl)uracil, and 0.1-50 micrograms/ml thymidine, thymine and 5-hydroxymethyluracil. Applicability of the method to determination of the kinetics of 5-ethyl-2'-deoxyuridine elimination by the isolated perfused rat liver was demonstrated; clearance of the drug was 1.29 ml/min.     

Effects of isoflavone compounds on the activation of phospholipase C.

The effect of isoflavone compounds, genistein and daidzein, on the breakdown of inositol phospholipids in 3T3 cells was studied. Genistein (100 micrograms/ml) inhibited the stimulation of the production of inositol phosphates by bombesin. The stimulated production of inositol phosphates by AlF-4 was also inhibited by genistein (IC50 = 0.6 micrograms/ml) and daidzein (IC50 = 2 micrograms/ml). However, the catalytic activity of phospholipase-C (PLC) in 3T3 cell extracts was not inhibited by these isoflavones. These results suggest that the isoflavones inhibited the activation of PLC at the G-protein or downstream of the sequences in signal transduction. In permeabilized 3T3 cells, the inhibition of AlF-4 plus adenosine triphosphate (ATP)-dependent PLC was recovered by increasing ATP but not AlF-4. Genistein also inhibited the activity of adenosine 5'-[3-O-thiotriphosphate] (ATP[S])-dependent PLC. The effect of genistein and other inhibitors of protein tyrosine kinases and phosphatases suggests that protein tyrosine phosphorylation is not involved in the activation of PLC in 3T3 cells and that AlF-4- and ATP[S]-mediated activation of PLC involves a different mechanism from the tyrosine kinase-mediated activation of PLC. Daidzein and genistein seem to interrupt the ATP-dependent step of PLC activation by a putative G-protein.     

Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures

Experimental data suggest that Resveratrol, a compound found in grapes and other fruits may influence cell proliferation and apoptosis. The aim of our experiments was to study the effect of Resveratrol on tumor cell cultures and an endothelial cell culture in order to examine the effect of various doses of this compound on active cell death and cell proliferation. Human tumor (HT-29, SW-620, HT-1080) and endothelial (HUV-EC-C) cells were treated with various doses of (0.1 to 100.0 microg/ml) Resveratrol in vitro. Cell number, apoptotic and mitotic index was measured 24, 48 and 72 h after treatment. Low doses (0.1-1.0 microg/ml) of Resveratrol enhance cell proliferation, higher doses (10.0-100.0 microg/ml) induce apoptosis and decrease mitotic activity, which is reflected in changes of cell number. Resveratrol influences dose dependently the proliferative and apoptotic activity of human tumor and endothelial cells. The possible role of formaldehyde in the mechanism of action of Resveratrol is discussed.     

Studies on thermophile products. V. Immunosuppressive profile in vitro of Bacillus stearothermophilus component, Fr.5-B.

The immunosuppressive profile of Bacillus stearothermophilus UK563 component, Fr.5-B, is presented in in vitro studies. Fr.5-B (0.1-1000 ng/ml), provided it was added at the initiation of mixed leukocyte reaction (MLR), inhibited dose-dependently the incorporation of tritiated thymidine ([3H]TdR) into mouse spleen cells and human peripheral blood lymphocytes. Even the addition of Fr.5-B 48 h after the onset of culture suppressed mouse MLR, unlike cyclosporin A (CYA). Fr.5-B significantly inhibited cytotoxic T lymphocyte generation determined by [3H]TdR-release micro-cytotoxicity assay by using mouse mastocytoma P815 as targets. Moreover, this component decreased dose-dependently the expression of class II major histocompatibility molecules (Ia) on mouse peritoneal macrophages induced by concanavalin A supernatant. The present results revealed the unique immunosuppressive property of Fr.5-B which was different from that of CYA.     

Proliferation of endothelial cells on the plasma-treated segmented-polyurethane surface: attempt of construction of a small caliber hybrid vascular graft and antithrombogenicity

To prepare a porous segmented-polyurethane (SPU) tube, a solution of SPU containing different concentrations of NaCl was coated on a glass rod and the coated SPU was immediately immersed in water. When the surface of the porous SPU, where bovine aortic endothelial cells are not normally capable of adhering and proliferating, was modified by plasma treatment, the proliferation of endothelial cells could be drastically improved. The cells proliferated confluently on the porous SPU surface prepared at low concentrations of NaCl below 10 g per 100 ml, but poorly on the porous surface prepared at high concentrations of NaCl. The construction of a hybrid vascular graft consisting of a porous SPU tube (2 mm in inner diameter, 5 cm in length) and endothelial cells was attempted. The cells cultured on the inner surface of the tube proliferated to confluency everywhere. From an in vitro antithrombogenic evaluation test, which involved the use of human blood, the present hybrid graft can be considered to provide an inert surface against thrombus formation and blood coagulation. Negligible changes in shape of human leukocytes in contact with bovine aortic endothelial cell surface occurred, suggesting that the bovine aortic endothelial cells used are immunologically less active against human blood.     

THYMIDINE UPTAKE, INCORPORATION AND DNA POLYMERASE ACTIVITY IN MURINE BLADDER TUMOR CELL,MBT–2, EXPOSED TO UV ACTIVATED DIHEMATOPORPHYRIN ETHER

Abstract— Photodynamic therapy (PDT) is a new modality for treatment of malignancy. In this paper, we reported the effect of UV activated dihematoporphyrin ether (DHE) on [³H] thymidine uptake and DNA synthesis in murine bladder tumor cells,MBT–2. Exponentially growing cells were pretreated with 0.05–5 μg/ml of DHE for 30 min in complete darkness prior to irradiation with 0.15-0.90 J/cm² of UV light (265 nm). The rates of thymidine uptake and DNA synthesis were suppressed in a DHE concentration and photic energy dependent manner. Double reciprocal analysis on the kinetics of the thymidine uptake and DNA synthesis indicated that the inhibition was non-competitive, i.e. decrease in both the apparent K^m value and maximum velocity in DHE plus UV light treated cells. The activities of DNA polymerase a and (3 were determined by [*H] dATP incorporation into DNA of permeabilizedMBT–2 cells. DNA polymerase a activity was approximately 60% of the control after 0.45 J/cm² of UV light exposure; a further inhibition of DNA polymerase a was observed when 0.5–5ng/W of DHE and UV photoradiation were combined. In contrast, a slight stimulation of DNA polymerase fJ was noted after a similar treatment. This study demonstrates that photodynamic therapy-induced suppression of DNA synthesis inMBT–2 cells is a complex process involving in reduction of thymidine transport as well as the perturbation of the enzymes involved in DNA synthesis.     

5-Hydroxymethyl-2'-deoxyuridine. Cytotoxicity and DNA incorporation studied by using a novel [2-14C]-derivative with normal and leukemic human hematopoietic cells

5-Hydroxymethyl-2'-deoxyuridine is a biologically active thymidine analogue. This investigation was aimed at characterizing the cytotoxicity of 5-hydroxymethyl-2'-deoxyuridine and its incorporation into DNA. Fifty percent inhibition of cellular proliferation, assessed by incorporation of [U-14C]-L-leucine in vitro, was caused by 1.7-5.8 X 10(-5) incorporation of [U-14C]-L-leucine in vitro, was caused by 1.7-5.8 X 10(-5) M 5-hydroxymethyl-2'-deoxyuridine in seven human leukemia cell lines. Higher concentrations of 5-hydroxymethyl-2'-deoxyuridine, i.e. 6-8 X 10(-5) M, were required for a comparable inhibition in human PHA-stimulated peripheral blood lymphocytes. A new synthesis procedure for [2-14C]5-hydroxymethyl-2'-deoxyuridine was developed. The net incorporation of [2-14C]5-hydroxymethyl-2'-deoxyuridine into DNA of hematopoietic cells was low. The possibility of a repair mechanism for 5-hydroxymethyluracil bound to DNA is discussed.     

EFFECTS OF ULTRAVIOLET LIGHT ON THYMIDINE INCORPORATION AND DNA CHAIN ELONGATION IN PHOTOREACTIVABLE INSECT CELLS

An insect cell line, IAL-PID2, was exposed to UV and analyzed for its ability to incorporate [3H]-thymidine and to elongate replicon-sized DNA fragments. After exposure to 5 or 10 J/m2 UV, the cells exhibited a rapid and prolonged depression in the rate of thymidine incorporation. Photoreactivation reduced this depression but did not entirely reverse it. For exposures of 5 J/m2 or above, full recovery did not occur until 18 h after exposure. The blockage of fork progression after UV exposure was fluence-dependent, with replication segments after exposure to 20 J/m2 being shorter than those observed after exposure to 10 J/m2. Immediately after exposure to either 10 or 20 J/m2, photoreactivation reversed blockage of fork progression, indicating that the (5-6) cyclobutyl pyrimidine dimer is responsible for blockage. This also indicates that blockage of fork progression may not be the only factor responsible for the prolonged depression seen in thymidine incorporation. Three hours after exposure to either 10 or 20 J/m2, replication segments were still significantly shorter than control segments. Photoreactivation completely reversed blockage after exposure to 10 J/m2, but did not completely reverse blockage after exposure to 20 J/m2, indicating that at such fluences, other lesions may play a role in UV-induced blockage of fork progression.     

Medium calcium concentration determines keratin intermediate filament density and distribution in immortalized cultured thymic epithelial cells (TECs).

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4-5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37 degrees C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 mug/ml), transferrin (10 mug/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.     

Metabolism of [2-14C]thymine and [2-14C]thymidine in germinating black gram (Phaseolus mungo) seeds

The metabolism of [2-14C]thymine and [2-14C]thymidine in the cotyledons and embryonic axes of black gram (Phaseolus mungo) seedlings was investigated. Both [2-14C]thymine and [2-14C]thymidine degraded extensively into [14C]CO2. The rate of release of [14C]CO2 from [2-14C]thymine was much greater than that from [2-14C]thymidine. Radioactivity from both precursors was also observed beta-ureidoisobutyric acid. This indicated that thymine was degraded by the reductive pathway of pyrimidine degradation. Small amounts of [2-14C]thymine and [2-14C]thymidine were salvaged for deoxyribonucleotide and DNA synthesis. The highest incorporation of [2-14C]thymine and [2-14C]thymidine into the DNA fraction was observed in 24 hour-old cotyledons where net DNA synthesis was not observed. These precursors seem to be utilised for DNA synthesis of organelles of the cotyledonary cells, probably mitochondria. In embronic axes, [2-14C]thymine is more effectively salvaged for DNA synthesis than [2-14C]thymine. The incorporation rate increased during the early phase of germination and attained its maximum at 48 h after which it decreased. No thymidine kinase activity was detected in either cotyledons or in the embryonic axes. Thymidine salvage seems to be catalysed by nucleoside phosphotransferase which is present both in the cotyledons and in the embryonic axes. This suggests that, in contrast to other pyrimidine and purine bases and nucleosides, no specific salvage system for thymine and thymidine is present in black gram seedlings.     

Pharmacokinetics of [6]-gingerol after intravenous administration in rats

A high-performance liquid chromatographic method to determine [6]-gingerol, a pungent constituent of ginger, in rat plasma was developed and a pharmacokinetic study was performed in rats. Quantitative analysis with high reproducibility was achieved for [6]-gingerol over the concentration range of 0.2-40 micrograms/ml. After bolus intravenous administration at a dose of 3 mg/kg, the plasma concentration-time curve was described by a two-compartment open model. [6]-Gingerol was rapidly cleared from plasma with a terminal half-life of 7.23 min and a total body clearance of 16.8 ml/min/kg. Serum protein binding of [6]-gingerol was 92.4%.     

Measurement of conjugated 1 beta-hydroxycholic acid in urine of newborns by specific radioimmunoassay.

Anti-tauro 1 beta-hydroxycholic acid antisera were prepared by immunizing rabbits with N-(1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholan-24-oyl)glycine bovine serum albumin conjugate. The immunoglobulin G fraction was obtained by ammonium sulfate precipitation, followed by diethylaminoethyl cellulose column chromatography. The antibody was characterized using [2-3H]tauro 1 beta-hydroxycholic acid which has a high affinity (Ka = 1.09 x 10(9) M-1) and reasonable specificity. Cross-reactivity for glyco 1 beta-hydroxycholic acid was 100% and those for other 1 beta-hydroxylated bile acids ranged from 4.32 to 29.6%. Concentrations of conjugated 1 beta-hydroxycholic acid in urine of newborns at 0-20 d after birth were determined by radioimmunoassay to be significant (0.2-11.1 micrograms/ml), exhibiting a tendency to increase during the 20 d after birth.     

Gas chromatographic assay for the new antitumor agent sulfamic acid diester (NSC 329680) and its stability in buffer, blood and plasma

A sensitive gas chromatographic assay with electron-capture detection has been developed for sulfamic acid diester (sulfamic acid 1,7-heptanediyl ester, NSC 329680) based on its conversion to 1,7-diiodoheptane in the presence of excess sodium iodide. The assay is linear up to 1 microgram/ml sulfamic acid diester and has a lower limit of detection of 25 ng/ml from 0.5 ml plasma. The coefficient of variation of the assay is 6.4% at 1 microgram/ml and 8.0% at 100 ng/ml. Sulfamic acid diester is relatively stable in 0.9% sodium chloride and 0.1 M sodium phosphate buffers, pH 7.0-9.0, with half-lives greater than 38 h. The major breakdown product of sulfamic acid diester is sulfamic acid 1,7-heptane-monoyl ester. When added to whole blood sulfamic acid diester shows concentration-dependent breakdown. At 50 and 100 micrograms/ml sulfamic acid diester, the half-time in whole blood is 6.9 h and 65% of the drug is sequestered by the blood cells. At 10 micrograms/ml sulfamic acid diester in blood, there is no detectable breakdown of the drug over 24 h and all of the drug is sequestered by the blood cells. Protein binding of sulfamic acid diester in human plasma is 82% at 10 micrograms/ml and 68% at 100 micrograms/ml.     

Understanding microchannel culture: parameters involved in soluble factor signaling

While the importance of autocrine-paracrine signaling in vivo is clear, the ability to study the effects of secreted endogenous factors in vitro is hampered by canonical culture platforms. In multi-well plates, the large air-liquid interface gives rise to convective flows that continually mix the fluid disrupting the local diffusion-based accumulation. Simple microchannels provide a more controlled microenvironment that can be used to study secreted factor effects. Here, we utilize microchannel culture to examine basic culture parameters and their interactions using normal mammary gland epithelial cells (NMuMG). The following parameters were studied: (1) cell density (80 vs. 240 cells mm(-2)), (2) exogenous growth factors (epidermal growth factor [EGF] vs. fetal bovine serum), (3) medium change frequency (1 h, 4 h, 12 h), and (4) culture platform (microchannels vs. 96-well plates). The cells exhibited increased growth rates in microchannels as compared to 96-well plates. Cell proliferation increased as the frequency of media change decreased. For the microchannel geometries used, important threshold concentrations were reached in a few hours. In aggregate, the results indicate that the function of the four factors and their interactions on NMuMG growth are spatially and temporally related by molecular diffusion in the controlled microchannel space. The convective-free microchannel environment may prove useful for studying soluble factor signaling in vitro, and to test models and predictions of autocrine-paracrine signaling.     

Combined chromatographic-isotopic dilution analysis of fecapentaenes in human feces

Fecapentaene-12 (FP-12) and fecapentaene-14 (FP-14) are genotoxic unsaturated ether lipids produced by colonic bacteria in man. We have developed and applied to feces collections from normal volunteers direct isotopic dilution procedures using tritium-labeled (at C5) FP-12 and FP-14 for measuring these compounds. FPs were recovered from feces by solvent extraction, silica cartridge clean-up, and analytical liquid chromatography. Low levels of FP-12 and FP-14 (less than 0.1 to 2.4 micrograms/g of freeze-dried feces) were observed. Identity of chromatographic peaks was established by co-elution and by ultraviolet absorption spectra obtained via photodiode array scanning. Two unknown peaks were tentatively identified from absorption spectra as closely related compounds with increased (hexane?) or decreased (tetraene?) number of double bonds. Levels of FPs increased after incubation of feces at 37 degrees C for 96 h under anaerobic conditions and pre-FP-12 and pre-FP-14 peaks were observed, which showed identical spectra with authentic FPs. These were interpreted to be isomeric forms of the all-trans-[3H]FPs used for the isotopic dilution analysis. Total FPs (including pre-FP) yielded a range of 0.3-80 micrograms FP-12 and 2.8-44 micrograms FP-14 per g of freeze-dried feces from the study group.     

Induction of A549 Nonsmall-Cell Lung Cancer Cells Proliferation by Photoreleased Nicotine

Caged compounds comprise the group of artificially synthesized, light-sensitive molecules that enable in situ derivation of biologically active constituents capable of affecting cells, tissues and/or biological processes upon exposure to light. Ruthenium-bispyridine (RuBi) complexes are photolyzed by biologically harmless visible light. In the present study, we show that RuBi-caged nicotine can be used as a source of free nicotine to induce proliferation of A549 nonsmall-cell lung cancer (NSCLC) cells by acting on nicotinic acetylcholine receptors expressed in these cells. RuBi-nicotine was photolyzed using LED light source with the spectrum matching RuBi-absorption. Photorelease of free nicotine ([Nic]_p/r) was quantified by high-performance liquid chromatography (HPLC). 5-s-long light exposure of 10 μm of RuBi-nicotine generated 2 μm [Nic]_p/r which enhanced A549 cell proliferation similarly to the 2 μm of plain nicotine during 72 h of cell culturing. Both RuBi-nicotine per se and its photolysis byproduct exerted no effect on A549 cells. We conclude that RuBi-nicotine can be a good source of free nicotine for inducing short- and long-term biological effects. Photolysis of RuBi-nicotine is quite effective, and can produce biologically relevant concentrations of nicotine at acceptable concentrations of the source material with the use of simple, inexpensive, and easily accessible light sources.