Surface plasmon excitation on a grating assisted by a cholesteric liquid crystal layer.

While surface plasmon excitation assisted by nematic liquid crystal layers has been comprehensively studied in different aspects, application of cholesteric structures for surface plasmon excitation remains an unexplored area. Moreover, structures including cholesteric layers and metal grating for surface plasmon excitation have never been considered, to the best of our knowledge. We studied theoretically and experimentally such structures focusing on different regimes of propagation of normal modes in the cholesteric liquid crystal layer. The application of such structures for sensing is accordingly discussed.

Electrospray ion beam deposition: soft-landing and fragmentation of functional molecules at solid surfaces.

The ion beam deposition (IBD) of rhodamine dye molecules on solid surfaces in high vacuum is explored in order to characterize the possibility of fabricating molecular coatings or nanostructures from nonvolatile molecules. Molecular ion beams with a well-defined composition are deposited on silicon oxide surfaces with a controlled kinetic energy. Photoluminescence spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) are employed in order to characterize the sample with respect to coverage, homogeneity, and the fraction of intact landed ions (soft-landing ratio). We find that homogeneous rhodamine films of defined composition can be produced at energies of 2-100 eV. The coverage is found to be proportional to the ion dose. Soft-landing is observed for energies up to 35 eV.

Magic alkali-fullerene compound clusters of extreme thermal stability.

The thermal stability of free pure C60-, as well as C60-alkali, and -alkaline-earth metal compound clusters is investigated. We find that small (C60)m-clusters (m

Fluorometric determination of 2'-beta-fluoro-2',3'-dideoxyadenosine 5'-triphosphate, the active metabolite of a new anti-human immunodeficiency virus drug, in human lymphocytes.

A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells.

A filter paper assay for hyaluronic acid synthetase: application to the enzyme from Swiss 3T3 fibroblasts.

An improved assay for hyaluronic acid (HA) synthetase is described that is suitable for rapid processing of large numbers of samples. High background levels of unincorporated radioactivity are removed by passage of the reaction through a Sephadex G-50 spin column. The labeled HA product is then precipitated onto glass fiber filters with cetylpyridinium chloride. Apparent Km values for HA synthetase from Swiss 3T3 fibroblasts are 10.8 and 58.4 microM for UDP-glucuronic acid and UDP-N-acetylglucosamine, respectively. HA synthetase activity of quiescent cells is 4.5% of that found in actively growing cells and is stimulated in response to 10% calf serum. There is a greater than 10-fold increase in HA synthetase activity when cells are harvested with hyaluronidase as compared with trypsin.

Cyclopentenyl uracil: an effective inhibitor of uridine salvage in vivo.

Cyclopentenyl uracil, a non-cytotoxic inhibitor of uridine kinase, was found to effectively block the salvage of circulating uridine by host and tumor tissues in the intact mouse. Dose-response characteristics of the inhibition were determined. Large doses (1 g/kg) of cyclopentenyl uracil were required, and the effect of a single dose fell rapidly over a 24-hr period. A sustained inhibition of uridine salvage of > 64-79% could be maintained by multiple doses of 1 g/kg given on an every 8-hr schedule. Mice given cyclopentenyl uracil (1 g/kg) every 8 hr for 5 days continued to gain weight and showed no signs of toxicity; however, the combination of cyclopentenyl uracil with a non-toxic dose of N-(phosphonacetyl)-L-aspartic acid (PALA; 200 mg/kg daily for 5 days) was lethal to mice, indicating that circulating uridine modifies the toxicity of agents that act on enzymes of the de novo pyrimidine pathway. Although the duration of action and potency of cyclopentenyl uracil are not ideal, this is the first demonstration of an effective inhibition of uridine salvage in the intact mouse with a non-cytotoxic agent. This makes possible the evaluation of concurrent inhibition of de novo and salvage routes to pyrimidine nucleotides as an approach to chemotherapy.

Non-steroidal anti-inflammatory drugs and tumor progression: inhibition of fibroblast hyaluronic acid production by indomethacin and mefenamic acid.

The antitumor effects of non-steroidal anti-inflammatory drugs (NSAIDs) have been documented in a variety of both clinical and experimental settings, although the mechanisms responsible remain unclear. In the present study, we show that the NSAIDs indomethacin and mefenamic acid inhibit the calf serum-stimulated production of hyaluronic acid (HA) in murine Swiss 3T3 fibroblasts, at concentrations where DNA synthesis is unaffected. HA is an extracellular matrix glycosaminoglycan associated with cell migration and tumor invasion. Our data suggest that one mechanism whereby NSAIDs inhibit tumor progression may be to inhibit the synthesis of HA by host fibroblasts, and that the eicosenoid pathway may represent an important control point in the growth-factor-mediated production of HA in fibroblasts. Thus the use of an agent which inhibits HA synthesis may be a novel approach to alter the invasive and metastatic properties of tumor cells in a non-cytotoxic fashion.

Inhibition of fibroblast hyaluronic acid production by suramin.

Hyaluronic acid (HA) is an extracellular matrix glycosaminoglycan localized in the stroma of solid tumors, where it facilitates cell movement and thus tumor invasion and metastasis. This localization of HA is due to its synthesis by stromal fibroblasts in response to paracrine factors produced by the tumor. Such tumor-stromal interactions have been shown to be crucial to the development and progression of prostate cancer. Suramin is an effective antitumor agent in hormone-refractory prostate cancer, but its mechanism(s) of action is not well understood. However, the properties of suramin as an agent which disrupts growth factor action, and the importance of tumor-stroma interactions in prostate tumor development and in HA synthesis led us to study the effect of suramin on HA synthesis. Suramin inhibited HA synthesis by calf serum-stimulated Swiss 3T3 fibroblasts at clinically relevant concentrations (IC50 = 183 micrograms/mL). Increasing the serum concentration from 10 to 20% did not change the IC50 for HA synthesis, but increased the IC50 for [3H]thymidine incorporation from 206 to 342 micrograms/mL, indicating that the antiproliferative effect of suramin can be dissociated from its effect on HA synthesis. Suramin did not alter the cellular concentrations of the two precursors for HA synthesis (UDP-glucuronic acid and UDP-N-acetylglucosamine) at early time points and did not inhibit the HA synthetase activity of isolated membranes at concentrations up to 800 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of de-novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo.

The relative contribution of de-novo and salvage synthesis to tissue pyrimidine nucleotide pools is an important parameter in the rational design of anti-pyrimidine therapies, but has not been measured in vivo. We have measured the contribution of de-novo synthesis to the total acid-soluble uracil nucleotide pool in mouse tissues by analysis of the incorporation of label after intra-peritoneal infusion of L-[15N]alanine. The contribution of salvage synthesis was measured by the incorporation of radiolabel after intravenous infusion of [14C]uridine. The results show that de-novo synthesis makes the larger contribution to the intestine uracil nucleotide pool, salvage synthesis makes the larger contribution to the kidney pool, and de-novo and salvage synthesis make roughly equal contributions to the liver pool. In tumors studied (L1210, P388, B16, Nettesheim), the contribution of de-novo synthesis was at least five times the contribution of salvage synthesis. The measurements were repeated 24 hours after a 400-mg/kg dose of N-phosphonacetyl-L-aspartic acid. De-novo synthesis was substantially inhibited in all tissues and tumors after this treatment, although significant residual activity was observed in the intestine and L1210 cells. Nettesheim carcinoma was the only tumor or tissue to show a significant increase in salvage synthesis after N-phosphonacetyl-L-aspartic acid treatment.

Uptake and metabolism of myo-inositol by L1210 leukaemia cells.

The initial rate of uptake of [3H]myo-inositol by L1210 murine leukaemia cells is directly proportional to the extracellular concentration and unaffected by several analogues of myo-inositol even at millimolar concentrations. Scyllitol, a geometric isomer of myo-inositol, partially inhibited the uptake of myo-inositol (40% at 0.1 mM). A portion of the uptake of myo-inositol was not inhibited even at 5 mM-scyllitol. At steady-state the intracellular concentration of [3H]myo-inositol is directly proportional to the extracellular concentration. Addition of myo-inositol to medium does not enhance the growth of L1210 cells; these cells can maintain an extracellular concentration of 20 microM-myo-inositol even when grown in myo-inositol-free medium. Synthesis of myo-inositol from glucose by L1210 cells was demonstrated by use of [13C]glucose and m.s. L1210 cells maintain myo-inositol pools by a combination of synthesis de novo and uptake of exogenous myo-inositol by either passive diffusion or a low affinity carrier.

Substrate properties of analogs of myo-inositol.

The hydrolysis of the minor cell membrane lipid phosphatidylinositol-4,5-bisphosphate mediates the action of many growth factors and hormones. As an approach to the development of specific inhibitors of this process, we have synthesized a series of analogs of myo-inositol and have evaluated their ability to serve as substrates for phosphatidylinositol synthetase. Modification at the 2-, 3-, or 4-positions produced compounds unable to serve as substrates, but several 5-modified analogs retained activity as substrates of phosphatidylinositol synthetase. The product formed from 5-deoxy-5-fluoro-myo-[3H]inositol by phatidylinositol synthetase was hydrolyzed by phospholipase D and gave 5-deoxy-5-fluoro-myo-inositol as the radiolabeled product. Two analogs, 5-deoxy-myo-inositol and 5-deoxy-5-fluoro-myo-inositol, were shown to permeate L1210 leukemia cells and be incorporated into cellular phospholipid. Analysis of the radiolabeled lipids formed on incubation of L1210 cells with 5-deoxy-5-fluoro-myo-[3H]inositol indicated that the fradulent lipid formed was further phosphorylated to the monophosphate but not to the diphosphate form.

D-myo-inositol (1,4)-bisphosphate 1-phosphate. Partial purification from rat liver and characterization.

A specific 1-phosphatase acting on myo-inositol (1,4)-biphosphate with a high affinity (Km = 0.9 microM) has been purified 49-fold from soluble proteins of rat liver by anion exchange chromatography followed by gel filtration. This enzyme has a molecular weight of 58,000 as estimated by gel filtration, a pH optimum of 7.5, and requires Mg++ for activity. The only product formed from myo-inositol (1,4)-bisphosphate is the 4-monophosphate. Of 7 other inositol phosphates examined only myo-inositol (1,3,4)-triphosphate was a substrate.

Antitumor activity and biochemical effects of cyclopentenyl cytosine in mice.

Cyclopentenyl cytosine, a recently synthesized inhibitor of cytidine 5'-triphosphate synthesis, has marked antitumor activity. Treatment with 1 mg/kg i.p. on days 1-9 following inoculation with tumor produced 111-122% increased median life span in mice bearing L1210 leukemia, 73-129% increased median life span in mice bearing P388 leukemia, and 58-62% increased median life span in mice with B16 melanoma. A subline of L1210 selected for resistance to 1-beta-D-arabinofuranosylcytosine was more sensitive to cyclopentenyl cytosine than the parent tumor line. L1210 cell growth in cultures was greatly inhibited (greater than 90%) by 0.1 microM cyclopentenyl cytosine, but cells were protected from the growth inhibitory effects by cytidine (20 microM) and to a lesser extent by uridine or deoxycytidine. Exposure of cultured L1210 cells to 1 microM cyclopentenyl cytosine inhibited formation of [3H]cytidine nucleotides from [3H]uridine by 30% during the first 15 min of exposure to drug and by greater than 95% after 2 h of exposure. Treatment of mice bearing L1210 ascites with cyclopentenyl cytosine (1 mg/kg) produced rapid depletion of cytidine nucleotide pools in the tumor cells; these pools fell to 35% of control within 30 min. The effects of cyclopentenyl cytosine on nucleotide pools were tissue selective; the cytidine nucleotide pools of spleen, liver, kidney, and intestine were less sensitive than that of the L1210 ascites tumor. Cytidine nucleotide pools of spleen and liver were depleted by higher doses (10 mg/kg) of cyclopentenyl cytosine.

The effect of 3-deazauridine and dipyridamole on uridine utilization by mice.

The inhibition of uridine utilization by 3-deazauridine, an inhibitor of uridine kinase, and by dipyridamole, an inhibitor of the facilitated transport of nucleosides was examined. 3-Deazauridine (500 mg/kg) markedly inhibited (greater than 70%) the formation of uracil nucleotides from uridine in liver, kidney, and L1210 tumor cells. The degree of inhibition is greatly reduced by 6 hr after administration of the drug. Dipyridamole (100 mg/kg) did not significantly reduce salvage of uridine by liver or kidney and produced only small, transient reductions in salvage by L1210 tumors. Dipyridamole pretreatment did not alter the rate of clearance of uridine from the plasma.

Inhibition of uridine kinase and the salvage of uridine by modified pyrimidine nucleosides.

Uridine kinase can play a crucial role in the provision of pyrimidine nucleotides for cellular nucleic acid synthesis, particularly when de novo synthesis is inhibited by chemotherapeutic agents. Therefore, uridine kinase is an attractive target for drug development. We examined a series of 29 analogs of uridine, most with modifications at the 5'-position, as inhibitors of uridine kinase in vitro and of uridine salvage by intact L1210 cells. Substitution at the 5'-position resulted in decreased efficacy as inhibitors of uridine kinase, particularly if the substituent was large. None of the analogs with 5'-position modifications effectively inhibited salvage of uridine by intact L1210 cells. Four carbocyclic pyrimidine nucleoside analogs (one series) were all effective competitive inhibitors of uridine kinase and of uridine salvage by intact L1210 cells. Cyclopentenyl uracil 19 shows promise for further development as it inhibits uridine salvage at nontoxic concentrations.

Salvage of circulating pyrimidine nucleosides by tissues of the mouse.

The metabolism of pyrimidine nucleosides present in the plasma of the mouse has been examined. Uridine and cytidine are rapidly cleared from the circulation with t1/2 of less than 5 min. Uracil, deoxycytidine, deoxyuridine, and thymidine are cleared more slowly with t1/2 of 9 to 13 min. Various tissues differed markedly in the extent of nucleotide formation from circulating nucleosides. Cytidine and uridine are predominantly converted to nucleotides (greater than 50%) rather than catabolized, whereas uracil is almost entirely degraded. Thymidine, deoxyuridine, and deoxycytidine are intermediate in the extent of their conversion to nucleotides: 8.9 to 21% of these nucleosides are salvaged in the mouse. Both anabolic and catabolic routes are important in the metabolism of pyrimidine nucleosides in vivo.