Effect of mimosine on DNA synthesis in mammalian cells

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.     

Evaluation of omeprazole and lansoprazole as inhibitors of cytochrome P450 isoforms

The human clearance of omeprazole and lansoprazole is conducted primarily by the hepatic cytochrome P450 (CYP) system. Efficacy data indicate few differences between these two drugs, but they may exhibit discrete drug interaction profiles. To compare the potency and specificity of these drugs as inhibitors of CYP isoforms, we performed in vitro studies with human liver microsomal preparations. Both drugs were potent, competitive inhibitors of CYP2C19, as measured by the conversion of S-mephenytoin to 4-hydroxymephenytoin (k(i) = 3.1 +/- 2.2 microM for omeprazole, K(i) = 3.2 +/- 1.3 microM for lansoprazole). For omeprazole, the highest concentration at which >70% inhibition of CYP2C19 was observed with no significant inhibitory effect on other isoforms was at least 20 times greater than K(i). Both drugs were competitive inhibitors of CYP2C9-catalyzed conversion of tolbutamide to 4-hydroxytolbutamide (K(i) = 40.1 +/- 14.8 microM for omeprazole, K(i) = 52.1 +/- 1.4 microM for lansoprazole) and were noncompetitive inhibitors of CYP3A-catalyzed conversion of dextromethorphan to 3-methoxymorphinan (K(i) = 84.4 +/- 4.0 microM for omeprazole, K(i) = 170.4 +/- 7.1 microM for lansoprazole). Lansoprazole was at least 5 times more potent (K(i) = 44.7 +/- 22.0 microM) than omeprazole (k(i) = 240.7 +/- 102.0 microM) as an inhibitor of CYP2D6-mediated conversion of dextromethorphan to dextrorphan. No inhibition of CYP1A2, assessed by measuring the conversion of phenacetin to acetaminophen, was noted. Our data suggest that whereas the inhibitory profiles of these two drugs are similar, lansoprazole may be the more important in vitro inhibitor of CYP2D6. Since its inhibition is very potent and has a broad "window of selectivity," omeprazole seems to be a useful, selective inhibitor of CYP2C19.     

Effect of ionizing radiation and topoisomerase II inhibitors on DNA synthesis in mammalian cells

We have used a novel, quantitative approach to study the effect of gamma-radiation and topoisomerase-II inhibitors on the initiation of DNA synthesis in eukaryotic cells. We found out that mild gamma-irradiation caused an almost immediate decrease in the rate of initiation of genomic DNA replication and stimulated DNA repair. This held true for two different cell lines. Ehrlich ascites tumor cells and Friend transformed erythroid cells, although the effect of gamma-radiation on Friend cells was more pronounced. At the same time, the synthesis of mitochondrial DNA was not affected by the irradiation. The effect of topoisomerase-II inhibitors on DNA initiation closely paralleled that of gamma irradiation, but did not stimulate repair. The fact that gamma-radiation and topoisomerase-II inhibitors, two types of agents that differ so profoundly, have practically the same effect on DNA synthesis speaks strongly in favour of the idea that eukaryotic cells have a general mechanism for coping with any disturbances in DNA integrity and chromatin structure. This mechanism is probably similar to the SOS-mechanism of prokaryotic cells and includes, as an early step, a slowdown of the initiation of replicative DNA synthesis.     

Comparison of the effect of lansoprazole and omeprazole on intragastric acidity and gastroesophageal reflux in patients with gastroesophageal reflux disease

A prospective study of the pharmacokinetics of itraconazole solution was performed in 11 patients who underwent allogeneic BMT (day of BMT = day 0) after a conditioning regimen including total body irradiation (TBI). Itraconazole solution (400 mg once a day) was given 7 days before BMT and continued up to the end of neutropenia unless another antifungal treatment was necessary. Blood samples were collected before itraconazole intake (Cmin) and 4 h later (Cmax) every other day for assays of itraconazole (ITRA) and its active metabolite hydroxy-itraconazole (OH-ITRA). The mean values of Cmin ITRA and OH-ITRA, respectively, were 287 +/- 109 ng/ml and 629 +/- 227 ng/ml at day -1 and 378 +/- 147 ng/ml and 725 +/- 242 ng/ml at day +1. The maximum Cmin values were observed at day +3. Six patients at day -1 (54%) and 8 at day +1 (72%) had satisfactory residual plasma concentrations of at least 250 ng/ml of unchanged ITRA. From day +1 to day +9, eight patients discontinued the itraconazole treatment, five of them had satisfactory plasma residual concentrations at this time. This work shows a good bioavailability of itraconazole oral solution during the early phase after allogeneic BMT, but more data are needed for the late phases.     

Influence of Ukrain on DNA, RNA and protein synthesis in malignant cells

3H labelled thymidine, uridine and leucine were used to evaluate the synthesis of DNA, RNA and proteins in malignant cells and normal cells incubated with Ukrain in different concentrations. Compared with the controls, the inhibiting effects of Ukrain are demonstrated in guinea pig hepatocytes, CIL hepatocytes, human tonsil cells, two murine lymphomas, murine myeloma, Yoshida cells, two HeLa strains, EsB- and EB- (murine) lymphomas. YAC-1, P815 and human WiDr cells. Ukrain inhibits the DNA, RNA and protein synthesis in malignant cell lines at relatively high concentrations and to a small extent in normal cells.     

Effect of split doses of N-methyl-N-nitrosourea on DNA repair synthesis in cultured mammalian cells

DNA repair was measured in human fibroblasts, mouse C3H 10 T 1/2 fibroblsts and rat hepatocytes by the non-semi-conservative incorporation of [3H]-TdR during DNA repair synthesis using liquid scintillation techniques. Confluent monolayers of these cells grown on cover slips were exposed to split doses (125 or 250 microgram/ml) of the mutagenic and carcinogenic alkylating agent MNU and DNA repair synthesis compared with that produced by a single dose (500 microgram/ml). No significant difference in DNA repair capacity was detected in the three cell lines treated with a single dose or split doses of MNU.     

Quercetin, a bioflavonoid, inhibits the DNA synthesis of human leukemia cells

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. It has been proposed that flavonoids may have potential as anticancer agents. To test an aspect of this hypothesis, we examined the effects of the flavonoid, quercetin, on the DNA synthesis of the human leukemia cell, HL-60. Quercetin induced a dose-dependent inhibition of DNA synthesis in the test range of 1 microM to 1 mM. The inhibitory effect on DNA synthesis was evident as early as 24 h after the addition of quercetin. At the concentrations of 10 microM, 100 microM and 1 mM, 50, 82 and 85% of DNA synthesis, respectively, was inhibited by quercetin as compared to the control. After 48 and 72 h incubation of the cells with 100 microM and 1 mM quercetin, DNA synthesis was almost completely abolished. These results suggest that the inhibitory effects of quercetin on HL-60 cell DNA synthesis is not due to a non-specific cytotoxic effect, since following removal of quercetin, the treated cells regrew normally.     

Effect of hydrocortisone on oxygen free radical generation by mononuclear cells

Corticosteroids are known to exert antiinflammatory and immunosuppressive effects. Since reactive oxygen species (ROS) induce tissue damage and inflammation and since mononuclear cells (MNCs) generate ROS, we investigated whether corticosteroids inhibit ROS generation by MNCs when given systemically. A single dose of either 300 mg (n = 8) or 100 mg (n = 6) of hydrocortisone (HC) was injected intravenously into eight and six subjects, respectively. Blood samples were obtained before and sequentially after the injection. Following 300 mg HC, N-formylmethionyl leucyl phenylalanine (fMLP)-induced ROS generation, assayed by measuring chemiluminescence with luminol, decreased significantly at 0.5 hours and reached a nadir at 2 hours (8% of basal, P < .001); thereafter, it gradually recovered, but was still below baseline at 24 hours. Following the dose of 100 mg HC, ROS generation decreased significantly at 1 hour (nadir, 30% of basal; P < .01) and gradually recovered to near basal level at 8 hours. Serum cortisol concentrations were markedly elevated over basal and remained elevated throughout the first 8 hours of the experiment, returning to baseline at 24 hours. This inhibition of ROS generation by HC (and other glucocorticoids) may have a role to play in mediating the antiinflammatory action of corticosteroids.     

Double-blind comparison of lansoprazole 15 mg, lansoprazole 30 mg, and placebo in the maintenance of healed gastric ulcer

Traditionally health care outcomes have been evaluated by measuring immediate and long-term survival. This paper demonstrates the importance of quality of life as an alternative outcome indicator by considering two dimensions of quality of life and how they affect patients following liver transplantation. These two dimensions are physical recovery and functional recovery and they are considered by analysing the literature exploring quality of life following liver transplantation. Physical recovery is be subdivided into sleep, pain and mobility and functional recovery is subdivided into occupational rehabilitation and domestic rehabilitation.     

Health-related quality of life outcomes of omeprazole versus ranitidine in poorly responsive symptomatic gastroesophageal reflux disease

OBJECTIVE: This study evaluated changes in health-related quality of life (HRQL) outcomes of once-daily omeprazole compared with ranitidine for the short-term treatment of patients with poorly responsive symptomatic gastroesophageal reflux disease (GERD). METHODS: A double-blind, randomized clinical trial, compared omeprazole versus ranitidine for the treatment of poorly responsive GERD. Eligible patients had a history of predominant heartburn symptoms with symptomatic heartburn after 6 weeks of ranitidine treatment. Patients were randomized to omeprazole 20 mg once daily (n = 156) or ranitidine 150 mg twice daily (n = 161) and followed for 8 weeks. Assessments were completed at baseline and after 8 weeks with physician-rated symptoms: Gastrointestinal Symptom Rating Scale (GSRS); Psychological General Well-Being (PGWB) Index; Sleep Scale; Impact on Daily Activities Scale, and Overall Treatment Effect. Primary HRQL endpoints were the GSRS reflux scale and PGWB total score. RESULTS: No differences between the 2 treatment groups were observed in baseline demographic, clinical or HRQL measures. After 8 weeks, omeprazole-treated patients had greater improvement in GSRS reflux scale scores (p<0.0001) and PGWB total scores (p = 0. 019) compared with ranitidine-treated patients. Significant between group differences favoring omeprazole were also observed in GSRS total scores (p<0.0001), abdominal pain scale scores (p = 0.003), and indigestion scale scores (p = 0.003), Impact on Daily Activities (p = 0.001), PGWB positive well-being (p = 0.015), anxiety (p = 0. 030), and general health scale scores (p = 0.010). Patient ratings of overall treatment effect demonstrated the significantly (p<0. 0001) greater benefits of omeprazole (mean = 5.26) compared with ranitidine treatment (mean = 3.83). CONCLUSIONS: Omeprazole treatment significantly reduced persistent reflux-related symptoms and normalized psychological well-being compared with ranitidine in poorly responsive symptomatic patients with GERD.     

IL-10 synthesis and secretion by peripheral blood mononuclear cells in haemodialysis patients

PURPOSE OF STUDY: IL-10 may explain the paradox between immunodeficiency and oversecretion of cytokines in chronic haemodialysis (HD) patients. We analysed the secretion of IL-10 by PBMC and the expression of IL-10 mRNA in 10 long-term HD patients (108-276 months), 10 short-term HD patients (3-18 months), and 10 healthy controls. RESULTS: Spontaneous IL-10 secretion was higher in HD patients than in controls (15 pg/ml vs 2 pg/ml, P = 0.004). It was detected in 13 of 20 patients and in 1 of 10 controls (P = 0.01). IL-10 mRNA expression was also higher in HD patients than in controls. Spontaneous secretions of IL-10 and IL-6 were positively correlated in patients. IL-10 secretion in response to LPS was higher than the upper limit of control range in 4 of 10 long-term HD patients and in no short-term HD patients (P = 0.04). IL-10 mRNA expression was also higher in long-term than in short-term HD patients. CONCLUSIONS: This study demonstrates that IL-10 is spontaneously synthesized and secreted in HD patients, supporting an immunomodulating role in this setting. The greater IL-10-producing capacity in long-term HD patients indicates a chronic effect of haemodialysis on PBMC responsiveness.     

Comparative effect of lansoprazole/amoxicillin with omeprazole/amoxicillin for the eradication of Helicobacter pylori in patients with duodenal ulcer

Lansoprazole, a potent antisecretory drug, possesses on an equimolar basis a 4-fold higher in vitro anti-Helicobacter pylori activity than omeprazole. In a prospective randomized study we compared lansoprazole 30 mg b.i.d. and amoxicillin 1 g b.i.d. with omeprazole 40 mg b.i.d. and amoxicillin 1 g b.i.d. for 14 days followed by lansoprazole 30 mg q.d. or omeprazole 20 mg q.d. for 14 additional days in 50 H. pylori positive duodenal ulcer patients (14f, 36m, age 27-83 [mean 43] years). H. pylori infection was diagnosed by histology (3 antral biopsies and 2 from gastric body, H & E- and Giemsa stain), rapid urease test (CLO) and culture in 39 patients, or by histology and rapid urease test in 11 patients. Control endoscopy was performed 4-6 weeks after the end of treatment. For eradication, a negative result in all 3 diagnostic modalities was required. The eradication rate was 43% (9/21 patients) in both treatment groups. 8 patients were lost to follow-up. The ulcer healing rate was 100% in both groups. Nonsmokers had a significantly higher (p = 0.026) eradication rate than smokers. No relevant adverse effects of the therapy occurred. 24 patients with persistent H. pylori infection were subsequently treated with lansoprazole 60 mg b.i.d. and amoxicillin 1 g b.i.d. for 14 days. Eradication was achieved in 5/22 (23%) patients (3/14 smokers, 2/8 nonsmokers), while 2 patients were lost to follow-up. 17 patients with persistent H. pylori infection after the second treatment received quadruple therapy consisting of metronidazole 500 mg t.i.d., tetracycline 500 mg q.i.d. bismuth-subcitrate 120 mg q.i.d. and lansoprazole 30 mg for 10 days. H. pylori eradication was achieved in 12/15 patients (80%). In conclusion, lansoprazole plus amoxicillin was equal to omeprazole plus amoxicillin in the treatment of H. pylori infected duodenal ulcer patients. Patients with eradication failure after dual therapy were successfully treated by quadruple therapy. In contrast, high dose lansoprazole and amoxicillin therapy was effective in only 23% of patients with persistent infection after standard dual therapy.     

The effects of retinol on in vitro immunoglobulin synthesis by cord blood and adult peripheral blood mononuclear cells

In this study we examined the effects of retinol (ROH), a metabolic precursor of retinoic acid (RA), on Staphylococcus aureus Cowan I (SAC)-induced immunoglobulin synthesis of cord blood mononuclear cells (CBMC) and adult peripheral blood mononuclear cells (PBMC). ROH augmented SAC-induced IgM synthesis of CBMC by 5.9 +/- 1.5-fold (n = 7, mean +/- s.d.), and IgG synthesis of adult PBMC by 16.3 +/- 5.1-fold (n = 3) at optimal concentrations of 10(-6) M and 10(-11) M, respectively. No augmenting effects could be demonstrated for the other immunoglobulin isotypes. Time-course studies showed that the synthesis of IgM by CBMC was accelerated with detectable immunoglobulin in supernatant fluids starting on day 3. ROH augmented immunoglobulin synthesis of CBMC stimulated by Epstein-Barr virus (EBV), a T cell-independent polyclonal activator, and of EBV-transformed B cell clones (2.5 +/- 0.2 and 4.1 +/- 1.5-fold increase, respectively), which suggests that ROH can act directly on B cells to enhance immunoglobulin synthesis. In contrast, when ROH was preincubated with cord blood T cells, washed and added to the B cell-enriched fraction with SAC, no increase (0.9-1.8-fold) in IgM synthesis was obtained. Thus, the principal mechanism(s) by which ROH augments immunoglobulin synthesis is by acting on B cells. This is in contrast to the immunoglobulin-enhancing effects of RA which is mediated by T cells, or T cell products, e.g. cytokine. Our studies suggest that RA and ROH may have different pathways of immunoglobulin-enhancing effects, perhaps mediated by different retinoid binding proteins resulting in gene activation and immunoglobulin synthesis.     

Effects of genistein and staurosporine on angiotensin II-induced DNA synthesis, protein synthesis and mitogen-activated protein kinase activation in vascular smooth muscle cells

This study examines the effects of protein kinase inhibitors and activator on angiotensin II-induced DNA synthesis and protein synthesis of rat aortic smooth muscle cells. In quiescent confluent cells, angiotensin II induced a concentration-dependent increase in thymidine incorporation and leucine incorporation. The tyrosine kinase inhibitor genistein caused an inhibition of the angiotensin II-induced DNA synthesis but not of the agent-induced protein synthesis. The protein kinase C inhibitors staurosporine and calphostin C caused an inhibition of the angiotensin II-induced protein synthesis but not of the agent-induced DNA synthesis. The protein kinase C activator phorbol 12-myristate 13-acetate stimulated protein synthesis. Angiotensin II stimulated mitogen-activated protein (MAP) kinases and the angiotensin II-induced MAP kinase activation was inhibited by genistein but not by staurosporine. These findings suggest that angiotensin II-induced DNA synthesis is at least partly mediated via protein-tyrosine phosphorylation and angiotensin II-induced protein synthesis is at least partly mediated by activation of protein kinase C. It seems likely that MAP kinase activation is involved in DNA synthesis but not in protein synthesis induced by angiotensin II.     

Comparison of the effect of two dose schedules of oral omeprazole with oral ranitidine on gastric aspirate pH and volume in patients undergoing elective surgery

We have compared gastric aspirate pH and volume at induction of anaesthesia in 222 patients who had received either omeprazole or ranitidine before elective operations. Omeprazole was given orally either as 40 mg on the evening before and 40 mg on the morning of surgery or as 80 mg on the morning of surgery. Ranitidine 150 mg was given orally on the evening before surgery and 2 h before anaesthesia. Treatment success was defined as aspirate pH > or = 2.5 and volume < 25 ml at induction of anaesthesia. Treatment was successful in 84% (95% confidence interval (CI) 73-91%) of patients in the omeprazole 40 + 40 mg group, 84% (95% CI 73-91%) in the ranitidine group and 73% (95% CI 61-83%) in the omeprazole 80 mg group. There were no statistically significant differences between the groups. Twelve patients in the omeprazole 80 mg group had gastric pH < 2.5 and four had volume > 25 ml. Only three patients had a gastric pH < 2.5 in the omeprazole 40 + 40 mg group and none had volume > 25 ml, which compared well with the ranitidine group. Omeprazole, given as 40 mg in the evening and 40 mg on the morning of operation, has a potential role for use in patients at risk for aspiration during general anaesthesia.     

Transdermal nicotine inhibits interleukin 2 synthesis by mononuclear cells derived from healthy volunteers

BACKGROUND: Smoking has either a beneficial or harmful effect on the course and recurrence of ulcerative colitis (UC) and Crohn's disease respectively. Transdermal application of nicotine had similar effects in UC and therefore was considered to be an effective basic drug that could be further developed in the search for new compounds in the treatment of acute exacerbations of corticosteroid-resistant UC. To clarify the hypothesis that nicotine exerts its anti-inflammatory effect in UC through selective inhibition of T-cell-derived cytokine synthesis, we studied in vivo effects of nicotine on cytokine production by human non-adherent mononuclear cells isolated from peripheral blood in a randomized, double-blind, placebo-controlled trial. METHODS: Healthy non-smoking volunteers applied for 2 weeks of nicotine patches (n = 12) with incremental doses of nicotine during the first week to achieve a maintenance dose of 15 mg per day, or placebo (n = 12). Blood was obtained before treatment and 1, 2, 3 and 6 weeks after the start of treatment. Cells were cultured in the absence or presence of phytohaemagglutinin for 48 h, and total amounts of interleukin 2 (IL-2), IL-4, IL-10, IL-13, interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) were measured. RESULTS: Transdermal nicotine caused a significant inhibition of IL-2 after 2 weeks' treatment compared with the placebo group. In addition, a diminished production of IL-10 and TNF-alpha in comparison with day 0 was observed. CONCLUSION: The beneficial effect of transdermal nicotine in ulcerative colitis may be mediated by a selective inhibition of the IL-2 production by mucosal mononuclear cells, which could result in diminished cell proliferation and consequently a reduction in the inflammatory process.     

Thymidine metabolism and DNA synthesis in Newcastle disease virus-infected cells

The inhibition of thymidine incorporation into DNA in Newcastle disease virus-infected cells has been studied. At 6 h after infection of L-929 cells at high multiplicity, transport of exogenous thymidine across the cell membrane was inhibited. The kinetics of this inhibition, decreased Vmax with no change in Km, suggest that there are fewer sites available for transport in infected cells. The conversion of thymidine to dTTP was not inhibited. Equilibrium of exogenous thymidine with the acid-soluble pool occurred more slowly and at a lower level of radioactivity than in uninfected cells, and there was a reduction in the rate of incorporation of exogenous thymidine into DNA. The reduction of incorporation into the pool and into DNA was proportionate. The size of total cellular dTTP pools was changed very little in infected cells. DNA synthesized in infected cells in the presence of [3H]BrdUrd had reduced incorporation of tritium but similar buoyant density to that from uninfected cells. The results show that Newcastle disease virus inhibits DNA synthesis directly and, in addition, decreases thymidine transport. Together these account for the overall decrease in thymidine incorporation into DNA of infected cells.