Protective effect and mechanism of Ginkgo biloba leaf extracts for Parkinson disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

AIM: To observe the effects of extracts of Ginkgo biloba leaves (EGb) on the Parkinson disease (PD) models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its ion 1-methyl-4-phenylpyridinium (MPP+). METHODS: MPTP was microinjected into substantia nigra of rats to induce a behavior change of rotation. EGb (ip, 50 or 100 mg.kg(1 . d-1) was pretreated consecutively for 19 d before MPTP administered and 1 d after MPTP administered. The contents of malondialdehyde (MDA), superoxide dismutase (SOD), and dopamine (DA) in substantia nigra of model rats were determined. Apoptosis of PC12 cells was induced by MPP+, and the protective effect of EGb (25, 50, and 100 mg/L) was also observed. The cells of apoptosis were observed under a microscope and counted under a fluoroscope after stained with AO/EB. RESULTS: EGb (100 mg . kg-1 . d-1) decreased the duration and frequency of the rotation of rats (P < 0.05, n = 10 ) while EGb (50 or 100 mg/L)inhibited the decreases of DA and SOD and the increase of MDA induced by MPTP, (P < 0.05 or P < 0.01, n = 10). MPP+ (10 micromol/L) induced the apoptosis of PC12 cells, and EGb (50 or 100 mg/L) prevented cells from apoptosis at 6 h, 12 h, and 24 h (P < 0.05 or P < 0.01, n = 3). CONCLUSION: EGb possesses protective effect on the PD models in vivo and in vitro. The anti-oxidation and anti-apoptosis may be one of the mechanisms underlying the neuroprotective effect of EGb.

Pilot study of a specific dietary supplement in tumor-bearing mice and in stage IIIB and IV non-small cell lung cancer patients.

Previously, a specific dietary supplement, selected vegetables (SV), was found to be associated with prolonged survival of stage III and IV non-small cell lung cancer (NSCLC) patients. In this study, several anticancer components in SV were measured; the anticancer activity of SV was assessed using a lung tumor model, line 1 in BALB/c mice. SV was also used in conjunction with conventional therapies by stage IIIB and IV NSCLC patients whose survival and clinical responses were evaluated. A daily portion (283 g) of SV was found to contain 63 mg of inositol hexaphosphate, 4.4 mg of daidzein, 2.6 mg of genistein, and 16 mg of coumestrol. Mouse food containing 5% SV (wt/wt) was associated with a 53-74% inhibition of tumor growth rate. Fourteen of the 18 patients who ingested SV daily for 2-46 months were included in the analyses; none showed evidence of toxicity. The first lead case remained tumor free for > 133 months; the second case showed complete regression of multiple brain lesions after using SV and radiotherapy. The median survival time of the remaining 12 patients was 33.5 months, and one-year survival was > 70%. The median survival time of the 16 "intent-to-treat" patients (including ineligible patients) was 20 months, and one-year survival was 55%. The Karnofsky performance status of eligible patients was 55 +/- 13 at entry but improved to 92 +/- 9 after use of SV for five months or longer (p < 0.01). Five patients had stable lesions for 30, 30, 20, 12, and 2 months; two of them, whose primary tumor was resected, used SV alone and demonstrated an objective response of their metastatic tumors. In addition to the two lead cases, eight patients had no new metastases after using SV. Three patients had complete regression of brain metastases after using radiotherapy and SV. In this study, daily ingestion of SV was associated with objective responses, prolonged survival, and attenuation of the normal pattern of progression of stage IIIB and IV NSCLC. A large randomized phase III clinical trial is needed to confirm the results observed in this pilot study.

[RP-HPLC method for determination of protopine in plasma and pharmacokinetics in rats].

AIM: To develop a reversed phase high performance liquid chromatographic method (RP-HPLC) for determination of protopine (Pro) in rat plasma and to investigate the pharmacokinetics of Pro in rats. METHODS: The column was packed with 5 microns C18. The mobile phase (pH 5.6) was a mixture of methanol-water-10% acetic acid (80:20:2). After twice extracted with ether under basic condition, and reextracted with 0.02 mol.L-1 sulfuric acid, protopine in the plasma samples was isolated well. The content of protopine in the plasma sample was measured by UV detector at 285 nm. RESULTS: The lowest limit of detection was 50 ng.mL-1. The intraday and interday precisions were 1.5%-3.0% and 2.1%-6.2%, respectively. The mean recovery was 80.6%-97.6%. A good linear relationship between the peak height and the concentration of protopine in rat plasma was observed. The pharmacokinetics of protopine had been investigated in rats after intravenous administration 10 mg.kg-1. The concentration-time curve of protopine in rat was confirmed to two-compartment open model. The T1/2 alpha, T1/2 beta, Ke, CL, Vd were 0.05 h, 1.85 h, 1.52 h, 6.41 L.h-1 and 17.27 L, respectively. CONCLUSION: This method is suitable for studies on pharmacokinetics of protopine.

Phase I/II study of stage III and IV non-small cell lung cancer patients taking a specific dietary supplement.

This phase I/II study evaluates the influence of selected vegetables (SV) that contain known antitumor components on the survival of stage III-IV non-small cell lung cancer (NSCLC) patients. All patients were treated with conventional therapies. SV was added to the daily diet of 5 stage I patients in the toxicity study group (TG) and 6 stage III and IV patients in the treatment group (SVG), but not to the diet of 13 stage III and IV patients in the control group (CG). Age, Karnofsky performance status (KPS), and body mass index of SVG and CG patients were comparable at entry. KPS declined in the CG patients (79 +/- 8 to 55 +/- 11) but improved in the SVG patients (75 +/- 8 to 80 +/- 13) one to three months after entry. Weight change in the CG, SVG, and TG patients was -12 +/- 5%, -2 +/- 2%, and +4 +/- 4%, respectively. The median survival time and mean survival of the CG patients were 4 and 4.8 months, but in the SVG patients they were 15.5 and 15 months (p < 0.01). No clinical signs of toxicity were found in the TG patients in the 24-month study period. Adding SV to the daily diet of NSCLC patients was found to be nontoxic and associated with improved weight maintenance, KPS, and survival of stage III and IV NSCLC patients.

Empirical and rational approaches for development of inhibitors of the human immunodeficiency virus--HIV-1.

The human immunodeficiency virus, HIV-1, is generally accepted to be responsible for AIDS. It is imperative that all approaches, empirical and rational, be taken for development of a drug for therapy of this disease. These approaches are discussed, with emphasis on the direction being pursued in our laboratory. Empirically, we found 3'-deoxy-2',3'-didehydrothymidine, a compound first synthesized for potential anticancer activity by J. Horwitz in the 1960s, to be a potent inhibitor of HIV-1. It is now in Phase II/III clinical trials. We have also synthesized several 2,5'-anhydro pyrimidine nucleoside analogs, which have interesting chemical and biological properties. We have evaluated a natural product, gossypol and synthesized various derivatives for anti-HIV-1 activity, but none were appreciably more inhibitory than the parent compound. More recently, we have taken the rational approach and synthesized a boron-modified tetrapeptide, Ac-Thr-Leu-Asn-boro-Phe, which corresponds to the COOH-terminal of the Phe-Pro scissle bond of the gag/pol gene polyprotein product. Potent inhibition of the HIV-1 encoded protease was observed. These approaches and findings will be discussed.

Enhancement of 5'-nucleotidase activity of human leukemic cells after fractionation: implications for cancer and aging.

Previous studies reported that 5'-nucleotidase activity was undetectable or at much lower levels in the homogenate of human chronic lymphocytic leukemic (CCL) cells than in normal lymphocytes. In the present study, 5'-nucleotidase specific activity in acute myelocytic leukemia (AML), which varied in a range from undetectable to 1.4 (nmoles/min.mg protein), was enhanced by cell fractionation, from undetectable in the homogenate, up to 18.8 +/- 1.2, 6.4 +/- 0.7 and 0.68 +/- 0.12 in plasma membranes, microsomes, and cytosol fraction, respectively. In a further fractionation of the cytosol of various leukemic cells with ammonium sulfate, 5'-nucleotidase specific activity increased up to 14-fold in the 60% (NH4)2SO4 fraction, with a recovery of 1266 +/- 115%. These data suggest that 5'-nucleotidase activity in fractionated leukemic cells is higher than reported previously and that the sum of 5'-nucleotidase activity in subcellular compartments is higher than that detected in the homogenate. Furthermore, even when 5'-nucleotidase was undetectable in a homogenate, it became detectable in the plasma membranes, suggesting that its ecto-enzyme function is still active in leukemic cells. The undetectable or low 5'-nucleotidase in the homogenate is indicative of (1) the enzyme itself being in an inactive form but becoming active after the fractionations, or (2) the presence of a factor(s) that prevents the enzyme from being detected but that is separated from the enzyme by the fractionations. In both cases, the rate of nucleotide catabolism by inactive 5'-nucleotidase in rapidly proliferating leukemic cells should be slower than when the enzyme is active. The present finding is consistent with our previous findings that during normal cell aging the high 5'-nucleotidase activity is associated with senescent non-proliferating cells but low or undetectable activity with rapidly proliferating immortal cells. The implications of 5'-nucleotidase for DNA synthesis in aging and cancer are discussed.

Implications of a 5'-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer.

5'-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56 degrees C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37 degrees C for 1 h and it was destroyed completely by heating at 100 degrees C for 30 min. When the heated (56 degrees C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of N-acetyl-beta-D-glucosaminidase or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5'-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35,000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggest that the previously reported undetectability of 5'-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5'-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5'-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.

A study of some mitochondrial and peroxisomal enzymes in human colonic adenocarcinoma.

Three mitochondrial enzymes, cytochrome oxidase, succinate dehydrogenase, and monoamine oxidase, and two peroxisomal enzymes, catalase and urate oxidase, were measured spectrophotometrically in the postnuclear supernatant prepared from homogenates of normal mucosa and carcinoma of the human colon. The specific activities, in both normal mucosa and carcinoma, varied from patient to patient. However, the difference in these activities between normal mucosa and carcinoma was consistent when patients were compared. The activities of cytochrome oxidase, succinate dehydrogenase, monoamine oxidase, and catalase were greater in normal mucosa than in carcinoma. In contrast, urate oxidase activity increased in carcinoma as compared with normal mucosa. Furthermore, cytochrome oxidase and succinate dehydrogenase decreased proportionally in carcinoma, supporting the concept that the mitochondrial respiration in normal tissue and carcinoma is quantitatively but no qualitatively changed. However, the decrease in monoamine oxidase activity in carcinoma was greater than that observed with other mitochondrial enzyme activities and was irregular. This suggests that a qualitative mitochondrial change may occur in carcinoma. In particular, the ratio between outer membrane enzyme activity and respiratory enzyme activity may be altered.

Oxidoreductase activities in normal rat liver, tumor-bearing rat liver, and hepatoma HC-252.

Experiments were carried out to determine if the difference in rates of cell proliferation between normal and neoplastic cells may be related to altered levels of oxidative enzymes. Assays were performed using homogenates from hepatocellular carcinoma HC-252, a rapidly growing and moderately well-differentiated tumor; from normal liver; and from the liver of the tumor-bearing ACI rat. Results of the mitochondrial enzymes indicated that the activities of cytochrome oxidase and succinate dehydrogenase were 3-fold lower in tumor homogenates than in liver homogenates. Monoamine oxidase activity could not be detected in HC-252; mixing experiments indicated no inhibitor was present in HC-252. Activities of th peroxisomal enzymes, urate oxidase, D-amino acid oxidase, and L-alpha-hydroxy acid oxidase were either undetected in the tumor or were 12-fold lower than in liver homogenates. The activity of xanthine oxidase, a cytoplasmic enzyme, was 5- to 6-fold lower in the tumor. Catalase activity in the tumor was also lower than in liver; this may be indicative of a lower oxidative environment at the cellular level. These enzyme activities of the liver of tumor-bearing rats were in the same range as those of normal rat liver, except that D-amino acid oxidase activity was slightly lower, and catalase activity was markedly lower and varied in a wide range. These results show an inverse correlation between the activities of oxygen-utilizing enzymes and rates of proliferation of one tumor line and its control. The possible implications of these results in neoplasia, cell proliferation, and cellular aging are discussed.

5'-Nucleotidase levels in normal and virus-transformed cells: implications for cellular aging in vitro.

5'-Nucleotidase activity in the postnuclear supernatant of normal and SV-40 virus-transformed human embryonic lung, chick embryo, and mouse embryo fibroblasts, WI-38, IMR-90, VA-13, CEF, 3T3, and SV3T3 cells was measured. In IMR-90 and CEF cells, both of which have a limited lifespan in vitro, 5'-nucleotidase activity increased 6- and 20-fold, respectively, with increasing population doublings. However, VA-13 cells, a permanent cell line, showed no increase in this enzyme activity with progressive population doublings. 5'-Nucleotidase activity was not detectable in two other permanent cell lines, 3T3 and SV3T3 cells. Of the six cell lines tested, the conspicuous enhancement in 5'-nucleotidase activity with increasing population doublings was observed only in normal cell lines and was absent in transformed cell lines. These observations suggest a molecular mechanism which may play a role in aging of normal cells in vitro and which may involve catabolism of nucleic acids.

Stabilization of electron spin resonance probes for photosynthesis studies.

The major obstacle to the study of functional/structural interrelationships of spinach chloroplasts by using spin labels has been the rapid loss of the electron paramagnetic resonance (EPR) signals upon illumination with visible light. The present study demonstrates that the addition of ferredoxin and NADP+ in the presence of N-tris(hydroxymethyl)methylglycine (Tricine) buffer at pH 7.1 or higher mitigates the rapid loss of Biradical X [N,N'-bis(1-oxyl - 2,2,5,5 - tetramethylpyrroline-3-carboxy)-1,2-diaminoethane] and Monradical A (2,2,5,5-tetramethyl-3-carbamidpyrroline-1-oxyl). However, the 5-line EPR spectrum characteristic of Biradical X in aqueous solution was changed to a dominantly 3-line spectrum within a few minutes after illumination in the presence of ferredoxin and NADP+. Analysis of the double integration of the first derivative EPR spectrum revealed no decrease in Biradical X concentration for more than 30 min of illumination. Our data suggest that Biradical X attaches to some soluble macromolecule(s) and that illumination of chloroplasts promotes such an attachment.