The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent.

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.

Doxorubicin pharmacokinetics following a single-dose infusion to sulphur-crested cockatoos (Cacatua galerita)

Objective To determine the pharmacokinetics of doxorubicin in sulphur-crested cockatoos, so that its use in clinical studies in birds can be considered. Design A pharmacokinetic study of doxorubicin, following a single intravenous (IV) infusion over 20 min, was performed in four healthy sulphur-crested cockatoos (Cacatua galerita). Procedure Birds were anaesthetised and both jugular veins were cannulated, one for doxorubicin infusion and the other for blood collection. Doxorubicin hydrochloride (2 mg/kg) in normal saline was infused IV over 20 min at a constant rate. Serial blood samples were collected for 96 h after initiation of the infusion. Plasma doxorubicin concentrations were assayed using an HPLC method involving ethyl acetate extraction, reverse-phase chromatography and fluorescence detection. The limit of quantification was 20 ng/mL. Established non-parametric methods were used for the analysis of plasma doxorubicin data. Results During the infusion the mean +/- SD for the C-max of doxorubicin was 4037 +/- 2577 ng/mL. Plasma concentrations declined biexponentially immediately after the infusion was ceased. There was considerable intersubject variability in all pharmacokinetic variables. The terminal (beta-phase) half-life was 41.4 +/- 18.5 min, the systemic clearance (Cl) was 45.7 +/- 18.0 mL/min/kg, the mean residence time (MRT) was 4.8 +/- 1.4 min, and the volume of distribution at steady state (V-SS) was 238 131 mL/kg. The extrapolated area under the curve (AUC(0-infinity)) was 950 +/- 677 ng/mL.h. The reduced metabolite, doxorubicinol, was detected in the plasma of all four parrots but could be quantified in only one bird with the profile suggesting formation rate-limited pharmacokinetics of doxorubicinol. Conclusions and clinical relevance Doxorubicin infusion in sulphur-crested cockatoos produced mild, transient inappetence. The volume of distribution per kilogram and terminal half-life were considerably smaller, but the clearance per kilogram was similar to or larger than reported in the dog, rat and humans. Traces of doxorubicinol, a metabolite of doxorubicin, were detected in the plasma.

Kidney side population reveals multilineage potential and renal functional capacity but also cellular heterogeneity

Side population (SP) cells in the adult kidney are proposed to represent a progenitor population. However, the size, origin, phenotype, and potential of the kidney SP has been controversial. In this study, the SP fraction of embryonic and adult kidneys represented 0.1 to 0.2% of the total viable cell population. The immunophenotype and the expression profile of kidney SP cells was distinct from that of bone marrow SP cells, suggesting that they are a resident nonhematopoietic cell population. Affymetrix expression profiling implicated a role for Notch signaling in kidney SP cells and was used to identify markers of kidney SP. Localization by in situ hybridization confirmed a primarily proximal tubule location, supporting the existence of a tubular niche, but also revealed considerable heterogeneity, including the presence of renal macrophages. Adult kidney SP cells demonstrated multilineage differentiation in vitro, whereas microinjection into mouse metanephroi showed that SP cells had a 3.5- to 13-fold greater potential to contribute to developing kidney than non-SP main population cells. However, although reintroduction of SP cells into an Adriamycin-nephropathy model reduced albuminuria:creatinine ratios, this was without significant tubular integration, suggesting a humoral role for SP cells in renal repair. The heterogeneity of the renal SP highlights the need for further fractionation to distinguish the cellular subpopulations that are responsible for the observed multilineage capacity and transdifferentiative and humoral activities.

Heat shock proteins in leukaemia cell differentiation and cell death

When HL60 cells were induced to differentiate to granulocyte-like cells with the agents N-methylformamide and tunicamycin an concentrations marginally below those which were cytotoxic, there was a decrease in the synthesis of the glucose- regulated proteins which preceded the expression of markers of a differentiated phenotype. There was a transient increase in the amount of hsp70 after 36 hours in NMF treated cells but in differentiated cells negligible amounts were detected. Inducers which were known to modulate hsp70 such as azetadine carboxylic acid did not induce differentiation suggesting early changes in the endoplasmic reticulum may be involved in the commitment to terminal differentiation of HL60 cells. These changes in group synthesis were not observed when K562 human chronic myelogenous leukemia cells were induced to differentiate to erythroid-like cells but there was a comparable increase in amounts of hsp70. When cells were treated with concentrations of drugs which brought about a loss in cell viability there was an early increase in the amount of hsp70 protein in the absence of any increase in synthesis. HL60 cells were treated with NMF (225mM), Adriamycin (1μM), or CB3717 (5μM) and there was an increase in the amounts of hsp70, in the absence of any new synthesis, which preceded any loss of membrane integrity and any significant changes in cell cycle but was concomitant with a later loss in viability of > 50% and a loss in proliferative potential. The amounts of hsp70 in the cell after treatment with any of the drugs was comparable to that obtained after a heat shock. Following a heat shock hsp70 was translocated from the cytoplasm to the nucleus, but treatment with toxic concentrations of drug caused hsp70 to remain localised in the cytoplasm. Changes in hsp70 turn-over was observed after a heat shock compared to NMF-treated cells. Morphological studies suggested that cells that had been treated with NMF and CB3717 were undergoing necrosis whereas the Adriamycin cells showed characteristics that were indicative of apoptosis. The data supports the hypothesis that an increase in amounts of hsp70 is an early marker of cell death.

An investigation of the effects of antitumour and other drugs on cell morphology and the cytoskeleton of erythrocytes

Human arythrocytes were used as a model system for an investigation of the mechanism of action of the antiproliferative drug Adriamycin. Erythrocytes were induced to undergo a change in morphology by elevation of intracellular calcium. It was revealed that the widely used media employed for the study of morphological change were unsuitable; a new incubation medium was developed so that cells were metabolically replete. In this medium echinocytosis took place both in a calcium concentration- and time-dependent manner. Pretreatment of erythrocytes with Adriamycin (10 M for 10 mins) protected the erythrocytes against calcium-induced echinocytosis at calcium concentrations < 150M. SDS-PAGE analysis of the cytoskeletal proteins prepared from erythrocytes revealed the calcium-induced proteolysis of two main cytoskeletal proteins: band 2:1 and band 4:1. Only the rate of the proteolysis of band 2.1 correlated with the onset of echinocytosis. Adriamycin inhibited the breakdown of band 2.1 even when the cells formed echinocytes; this raises doubts concerning the importance of band 2.1 in the maintenance of discocyte morphology. Adriamycin only marginally inhibited the purified calcium-activated thio protease (calpain). Calcium-loading of human erythrocytes increased the phosphorylation of several major cytoskeletal proteins including pp120, band 3, band 4.1 and band 4.9. The pattern of increase resembled that induced by 12-0-tetradecanoyl-phorbol-13-acetate. Pre-treatment with Adriamycin prior to calcium loading caused a general lowering of basal phosphorylation. Adriamycin had no effect on the activity of the calcium-activated phospholipid-dependent protein kinase (protein kinase C). A hypothesis is put forward that the morphological transition of erythrocytes might be dependent upon the activity of a contractile system.

La place de la chimiothérapie dans le traitement du cancer broncho pulmonaire

Single agent chemotherapy of advanced lung cancer is still unsatisfactory. The most encouraging results have been obtained with high dose intermittent treatment with cyclophosphamide, methotrexate or adriamycin. Combination chemotherapy is probably more efficacious than single agent chemotherapy, especially in oat cell carcinoma. The best treatment schedules give a response rate of approximately 80%. The value of adjuvant chemotherapy is not yet established, but new controlled clinical trials are indicated considering the poor results achieved by surgery and radiotherapy.

Expression and Role of CD166 in the Chronic Kidney Disease

Background: CD166, an adhesion molecule of the immunoglobulin superfamily, is one of the crucial effectors that traffic lymphocytes into tissues. Till now, the expression and role of CD166 in the chronic kidney disease remains unknown. Objectives: In the present study, we are to examine the expression of CD166 in the chronic kidney disease, and to explore its function with CD4+ T cells. Materials and Methods: CD166 expression was tested by Flow Cytometry (FACS) in the primary macrophages stimulated with LPS. In vivo, the expression of CD166 and CD4 were examined in the kidney tissues of adriamycin-induced nephropathy (AN) mice by immnohistochemistry. Macrophages and lymphocytes were co-cultured, the interaction between CD166 and CD4 was tested by immunofluorescent staining. Furthermore, the effects of CD166 on the activation and proliferation of T cells were explored. Results: In this study, CD166 expression was found to be upregulated on activated macrophages and glomerular endothelia in the adriamycin-induced nephropathy (AN) mice and CD4+ T cells were increased with CD166 expression in the AN mice. The interaction between macrophages and CD4+ T cells indicated that CD166 played a key role in the recruitment of lymphocytes in the chronic kidney disease, and neither proliferation nor activation of T cells was affected by CD166. Conclusions: CD166 expressed on macrophages and endothelia in AN kidney, and the function was related to the recruitment of CD4+ T cells into inflamed kidney, indicating that CD166 may be a potential target for reducing the inflammatory infiltrates in the chronic kidney disease.

Design, synthesis and antiproliferative activity of hydroxyacetamide derivatives against HeLa cervical carcinoma cell and breast cancer cell line

Purpose: To design and develop a new series of histone deacetylase inhibitors (FP1 - FP12) and evaluate their inhibitory activity against hydroxyacetamide (HDAC) enzyme mixture-derived HeLa cervical carcinoma cell and MCF-7. Methods: The designed molecules (FP1 - FP12) were docked using AUTODOCK 1.4.6. FP3 and FP8 showed higher interaction comparable to the prototypical HDACI. The designed series of 2-[[(3- Phenyl/substituted Phenyl-[4-{(4-(substituted phenyl)ethylidine-2-Phenyl-1,3-Imidazol-5-One}](-4H- 1,2,4-triazol-5-yl)sulfanyl]-N-hydroxyacetamide derivatives (FP1-FP12) was synthesized by merging 2- [(4-amino-3-phenyl-4H- 1, 2, 4-triazol-5-yl) sulfanyl]-N-hydroxyacetamide and 2-{[4-amino-3-(2- hydroxyphenyl)-4H-1,2, 4-triazol-5-yl]sulfanyl}-N hydroxyacetamide derivatives with aromatic substituted oxazolone. The biological activity of the synthesized molecule (FP1-FP12) was evaluated against HDAC enzyme mixture-derived HeLa cervical carcinoma cell and breast cancer cell line (MCF-7). Results: HDAC inhibitory activity of FP10 showed higher IC50 (half-maximal concentration inhibitory activity) of 0.09 μM, whereas standard SAHA molecule showed IC50 of 0.057 μM. On the other hand, FP9 exhibited higher GI50 (50 % of maximal concentration that inhibited cell proliferation) of 22.8 μM against MCF-7 cell line, compared with the standard, adriamycin, with GI50 of (-) 50.2 μM. Conclusion: Synthesis, spectral characterization, and evaluation of HDAC inhibition activity and in vitro anticancer evaluation of novel hydroxyacetamide derivatives against MCF-7 cell line have been achieved. The findings indicate the emergence of potentialanticancer compounds.