Ecdysteroid signaling in ecdysteroid-resistant cell lines from the polyphagous noctuid pest Spodoptera exigua

Although dibenzoylhydrazine-type non-steroidal ecdysone agonists such as methoxyfenozide (RH-2485) have an excellent performance record, the emergence of resistance could severely compromise the efficacy of these compounds in integrated pest management programs. To investigate possible mechanisms of resistance, cell lines derived from the polyphagous noctuid pest Spodoptera exigua (Se4 cells) were selected for continuous growth in the presence of high concentrations of 20-hydroxyecdysone (20E) or methoxyfenozide. Here we describe an analysis of ecdysteroid receptor signaling in the ecdysteroid-resistant Se4 cell lines. In contrast to other ecdysteroid-resistant cell lines described in literature, our data support the existence of a normal functioning ecdysteroid receptor complex in the resistant Se4 cell lines: (1) using a recombinant BmNPV baculovirus as a transduction tool, activation of an ecdysone-responsive luciferase cassette was demonstrated; (2) the early gene HR3 is constitutively expressed in the resistant cell lines that are grown in the presence of 20E or methoxyfenozide. Quantitative RT-PCR experiments indicated that expression levels of SeEcR mRNA were comparable among sensitive and resistant cell lines. Sequencing of PCR fragments also revealed the presence of SeEcR mRNA with a wild-type ligand-binding domain in resistant cells. Finally, a possible role for the gene FTZ-F1, whose expression correlates with the absence of circulating ecdysteroids during insect development, in the resistance mechanism was investigated. However, it was observed that FTZ-F1, in contrast to what is observed during insect development, is constitutively expressed in Se4 cells and that its expression is not regulated by the addition of ecdysteroid. It is proposed that the resistance mechanism in Se4 cells resides at the coupling between the conserved hierarchical cascade of early and early-late gene expression and the differentiation program in the Se4 cell line. The use of insect cell lines for the investigation of resistance against dibenzoylhydrazine ecdysone agonists and their relevance for uncovering resistance mechanisms in insects during pest control programs is discussed.     

Ecdysteroid resistant subclones of the epithelial cell line from Chironomus Tentans (insecta, diptera). I. Selection and characterization of resistant clones

Summary Chironomus tentans cells were cultured in the presence of gradually increasing concentrations of 20-OH-ecdysone or a nonsteroidal molting hormone agonist, the benzoylhydrazine RH 5992, for a period of about 2 yr. From these cultures, subclones were selected, which are resistant to up to 25 μM 20-OH-ecdysone according to morphological (changes in cell shape and cell arrangement) and physiological criteria (acetylcholinesterase induction, secretion of chitinolytic enzymes, thymidine incorporation). Some subclones, selected in the presence of 20-OH-ecdysone, are resistant only to molting hormone, but still respond to RH 5992 morphologically and biochemically, whereas subclones selected in the presence of the benzoylhydrazine showed no reaction neither to 20-OH-ecdysone nor to the hormone agonist. Hormone resistance is stable; 3 mo. after hormone withdrawal, resistant clones still do not respond to renewed exposure to 20-OH-ecdysone or RH 5992, respectively. Because in all resistant subclones tested so far all hormonally regulated responses known from sensitive cells were no longer detectable, it is assumed that the hormone signaling pathway itself is interrupted. Possible mechanisms of hormone resistance were discussed.     

Liposomal daunorubicin overcomes drug resistance in human breast,ovarian and lung carcinoma cells

Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC50 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC50 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC50 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC50 of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC50 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC50 237 nM) and H69VP small-cell lung carcinoma cells (IC50 27 nM). Empty liposomes did not affect the IC50 for free DR in the three resistant cell lines, nor did empty liposomes affect the IC50 for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.     

Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.

Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.     

The effect of doxorubicin and retinoids on proliferation, necrosis and apoptosis in MCF-7 breast cancer cells

Doxorubicin (Adriamycin) is the most active drug in the treatment of breast cancer. The aim of this study was to investigate the interaction of doxorubicin and retinoids in the inhibition of proliferation of hormone sensitive (ER+) human breast cancer cell line MCF-7 and to find out whether this combination can result in the enhancement of its therapeutic effect. As a comparison we also used estradiol and tamoxifen. We also made an attempt to elucidate the effect of these compounds on the stimulation of the apoptotic pathway in breast cancer cells. Cell proliferation in a 24-hour culture was assessed by [3H] thymidine incorporation into cancer cells and by immunocytochemical analysis of cellular cycle-related PCNA and Ki-67 antigens expression, after the incubation of the cell culture with 10, 20 and 50 nM doxorubicin (DOX), 2 nM estradiol (E2), 10 microM tamoxifen (TAM) and 1 nM, 0.01, 0.1, 1 and 10 microM of all-trans retinoid acid (ATRA). The assessment of cell viability and analysis of apoptotic and necrotic cells were performed after the 72-hour incubation of the culture with the examined substances and following apoptosis induction using acridine orange and ethidine bromide. Of the doxorubicin concentrations used in the study, 20 nM inhibited thymidine incorporation to 84.83 +/- 10.00% (control=100%). In the same culture conditions, 2 nM E2 stimulated cancer cells to 157.09 +/- 8.84%. Concentrations of 10 microM TAM and 10 microM ATRA inhibited the proliferation to 63.16 +/- 7.85% and 52.19 +/- 3.21%, respectively. A statistically significant reduction of these values was observed when 20 nM DOX was added to medium with E2 - 39.24 +/- 7.6%, TAM - 48.34 +/- 2.05% and ATRA - 21.98 +/- 1.69%, respectively; the percentage of PCNA- and Ki-67-positive cells was also reduced. Despite high antiproliferative efficacy of 20 nM DOX and 10 microM ATRA combination, the percentage of apoptotic cells was only 25 +/- 0.81%, being similar to that obtained in the culture with 20 nM DOX. The concentrations of 10, 20 and 50 nM DOX that were used to inhibit the proliferation of MCF-7 cell line were not particulary effective. The inhibitory effect was obtained when 20 nM of DOX and E2, TAM or ATRA were used simultaneously. The use of E2 caused a two-fold decrease in the percentage of proliferating cells. It was also shown that the effectiveness of DOX in combination with ATRA is significantly higher than that of DOX combined with TAM, which might suggest a valuable approach to the treatment of breast cancer.     

Effects of Ca2+-channel antagonists on nucleoside and nucleobase transport in human erythrocytes and cultured mammalian cells

Lidoflazine strongly inhibited the equilibrium exchange of uridine in human erythrocytes (Ki approximately 16 nM). Uridine zero-trans influx was similarly inhibited by lidoflazine in cultured HeLa cells (IC50 approximately to 80 nM), whereas P388 mouse leukemia and Novikoff rat hepatoma cells were three orders of magnitude more resistant (IC50 greater than 50 microM). Uridine transport was also inhibited by nifedipine, verapamil, diltiazem, prenylamine and trifluoperazine, but only at similarly high concentrations in both human erythrocytes and the cell lines. IC50 values ranged from about 10 microM for nifedipine and about 20 microM for verapamil to more than 100 microM for diltiazem, prenylamine and trifluoperazine. The concentrations required for inhibition of nucleoside transport are several orders higher than those blocking Ca2+ channels. Lidoflazine competitively inhibited the binding of nitrobenzylthioinosine to high-affinity sites in human erythrocytes, but did not inhibit the dissociation of nitrobenzylthioinosine from these sites on the transporter as is observed with dipyridamole and dilazep.     

LIPOSOMAL DAUNORUBICIN OVERCOMES DRUG RESISTANCE IN HUMAN BREAST,OVARIAN AND LUNG CARCINOMA CELLS

ABSTRACT

Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC₅₀ 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC₅₀ 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC₅₀ 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC₅₀ of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC₅₀ 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC₅₀ 237 nM) and H69VP small-cell lung carcinoma cells (IC₅₀ 27 nM). Empty liposomes did not affect the IC₅₀ for free DR in the three resistant cell lines, nor did empty liposomes affect the IC₅₀ for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.     

Localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane

Changes in the localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane were studied. A 2 h treatment of excised root with valinomycin (20 microM), N,N-dicyclohexylcarbodiimid (100 microM), gramicidin S (20 microM) and chlorpromazine (100 microM) caused an increased loss of potassium by cells, a decreased respiration and changes in the localization of ATPase activity and in cell ultrastructure. Differences in the observed changes may be conditioned by different mechanisms of action of the membrane active compounds used. It is concluded that changes in the localization of ATPase activity and ultrastructure may indicate some early specific responses of root cells, whereas the increase in the ion conductivity and decrease in respiration under disruption of ion homeostasis caused by membrane active compounds indicate unspecific responses of cells.     

Intestinal absorption of oestrone, oestrone glucuronide and oestrone sulphate in the rat in situ--II. Studies with the Doluisio technique

The absorption of oestrone (E1), oestrone glucuronide (E1G) and oestrone sulphate (E1S) from the small intestine of anaesthetized rats has been evaluated using the Doluisio in situ technique. Luminal disappearance of E1 was biphasic, which is consistent with a 3-compartment model; t1/2 alpha (first phase) was less than 5 min and t1/2 beta (second phase) approx 27 min for each concentration of steroid studied (trace identical to 10 nM, 1 microM and 10 microM). In contrast, luminal disappearance of E1G and E1S was monoexponential; t1/2 for E1G was 159, 229 and 299 min (trace identical to 200 nM, 10 microM and 100 microM respectively) and for E1S, 215, 174 and 192 min (trace identical to 10 nM, 10 microM and 100 microM respectively). There was a good correlation between the luminal disappearance data and recovery of steroid in bile. Adsorption of E1S was estimated from the initial rapid fall in luminal content within the first 5 min after drug administration. The study provides further evidence that E1S can be absorbed intact. Since saccharolactone only caused a reduction in E1G absorption of 32% we also conclude that part of the administered E1G was absorbed intact.     

Isolation of a taxol-resistant Leishmania donovani promastigote mutant that exhibits a multidrug-resistant phenotype

We raised a strain of Leishmania donovani in the laboratory that was resistant to 500 nM taxol. The IC50 of the wild-type strain for taxol was 35 nM and that of the taxol-resistant strain (T-500) was 1 microM. The T-500 strain exhibited a Mdr phenotype; it was also resistant to other unrelated drugs like vinblastine, adriamycin and the commonly used antimonial drugs pentostam and glucantime. Verapamil (20 nM), a calcium channel blocker, was found to reverse the resistance of T-500 to taxol. Acquired resistance to taxol has been reported to be mediated by alterations involving tubulin in cancer cells. Thus polymerisation assays with tubulin fractions in wild-type versus taxol-resistant cells (T-500) were performed in vitro. The tubulin fraction from T-500 was more resistant to in vitro polymerisation than the tubulin isolated from the wild-type, suggesting that this is one means by which the parasite may acquire resistance to taxol.     

Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes

The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATP-dependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 microM and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol x min(-1) x mg MRP2(-1) at 100 nM LTC4. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 microM. Our data document the first reconstitution of transport-active purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but co-reconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC4 transport function of MRP2 is not dependent on calnexin. The purified, transport-active MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.     

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor G?-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or G?-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.     

Oxadiazole derivatives as a novel class of antimitotic agents: Synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines

Oxadiazole derivatives were synthesized and evaluated for their ability to inhibit tubulin polymerization and to cause mitotic arrest in tumor cells. The most potent compounds inhibited tubulin polymerization at concentrations below 1 microM. Lead analogs caused mitotic arrest of A431 human epidermoid cells and cells derived from multi-drug resistant tumors (10, EC(50)=7.8 nM). Competition for the colchicine binding site and pharmacokinetic properties of selected potent compounds were also investigated and are reported herein, along with structure-activity relationships for this novel series of antimitotic agents.     

Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-ferrocene derivatives

Novel derivatives bearing a ferrocene attached via a piperazine linker to C-10 of the artemisinin nucleus were prepared from dihydroartemisinin and screened against chloroquine (CQ) sensitive NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf) parasites. The overall aim is to imprint oxidant (from the artemisinin) and redox (from the ferrocene) activities. In a preliminary assessment, these compounds were shown to possess activities in the low nM range with the most active being compound 6 with IC₅₀ values of 2.79?nM against Pf K1 and 3.2?nM against Pf W2. Overall the resistance indices indicate that the compounds have a low potential for cross resistance. Cytotoxicities were determined with Hek293 human embryonic kidney cells and activities against proliferating cells were assessed against A375 human malignant melanoma cells. The selectivity indices of the amino-artemisinin ferrocene derivatives indicate there is overall an appreciably higher selectivity towards the malaria parasite than mammalian cells.     

17Alpha-alkan (or alkyn) amide derivatives of estradiol as inhibitors of steroid-sulfatase activity

To develop inhibitors of steroid sulfatase without residual estrogenic activity, we have designed a series of estradiol (E2) derivatives bearing an alkan (or alkyn) amide side chain at position 17alpha. A hydrophobic alkyl group was selected from our previous study where 17alpha-octyl-E2 was found to inhibit strongly the steroid-sulfatase activity. Furthermore, it is known that an alkylamide side chain blocks the estrogen-receptor activation. Starting from ethynylestradiol, the chemical synthesis of target compounds was short and efficient with overall yields of 22-42% (3 or 4 steps). Among these compounds, N-octyl,N-methyl-3-(3',17'beta-dihydroxy-1',3',5'(10')-estratrien- 17'alpha-yl)-propanamide (15) was the most potent inhibitor, with an IC50 value of 0.08 microM for the transformation of estrone sulfate (E1S) to estrone (E1) by homogenated JEG-3 cells. N-butyl, N-hexyl, and N,N-dioctyl propanamide derivatives of E2 (IC50 values of 6.4, 2.8, and >20 microM, respectively) were less potent inhibitors than N-octyl analog 15. Furthermore, the unsaturated propynamide analog of 15 gave lower inhibition (four times) than the saturated compound. Compound 15 is also about 100-fold more effective in interacting with the enzyme than substrate E1S itself. The ability of target compounds to bind the estrogen receptor, to stimulate the proliferation of estrogen-sensitive ZR-75-1 cells, or to inhibit the E2-stimulation of ZR-75-1 cells was also evaluated. Although a mixed estrogenic/anti-estrogenic activity was obtained for tested compounds at 1 microM, no estrogenic activity was observed at 0.03 microM for 15. In conclusion, a promising inhibitor of steroid-sulfatase activity was obtained by introducing a hydrophobic octyl group in a 17alpha-propanamide side chain of E2, but further structure-activity relationships (SAR) studies are necessary to minimize the residual estrogenic activity.     

Differing sensitivity of tumor cells to apoptosis induced by iron deprivation in vitro

We studied the sensitivity of tumor cells to the induction of apoptosis by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Mouse 38C13 cells and human Raji cells die within 48 and 96 h of incubation in iron-deficient medium, respectively. On the contrary, mouse EL4 cells and human HeLa cells are completely resistant to the induction of death under the same experimental arrangement. Deoxyribonucleic acid fragmentation analysis by agarose gel electrophoresis as well as flow cytometric analysis after propidium iodide staining detected in 38C13 and Raji cells, but not in EL4 and HeLa cells, changes characteristic to apoptosis. The 38C13 cells, sensitive to iron deprivation, also displayed a similar degree of sensitivity to apoptosis induction by thiol deprivation (achieved by 2-mercaptoethanol withdrawal from the culture medium) as well as by rotenone (50 nM), hydroxyurea (50 microM), methotrexate (20 nM), and doxorubicin (100 nM). Raji cells shared with 38C13 cells a sensitivity to rotenone, methotrexate, doxorubicin, and, to a certain degree, to hydroxyurea. However, Raji cells were completely resistant to thiol deprivation. EI4 and HeLa cells, resistant to iron deprivation, also displayed a greater degree of resistance to most of the other apoptotic stimuli than did their sensitive counterparts. We conclude that some tumor cells in vitro are sensitive to apoptosis induction by iron deprivation, while other tumor cells are resistant. All the tumors found to be sensitive to iron deprivation in this study (four cell lines) are of hematopoietic origin. The mechanism of resistance to apoptosis induction by iron deprivation differs from the mechanism of resistance to thiol deprivation.     

Antennal responses of Cydia pomonella (L.) exposed to surfaces treated with methoxyfenozide

Electroantennogram (EAG) measurements were recorded from the antennae of male and female codling moth, Cydia pomonella L., to determine whether adult moths exposed to surfaces treated with the ecdysteroid agonist methoxyfenozide experience a decline in their antennal reception and thus olfactory sensitivity. Such a phenomenon would offer a possible mechanism for the previously reported decreased responsiveness from moths treated with methoxyfenozide to pheromone‐ and plant volatile‐based monitoring lures. Mean EAG data revealed that the antennae from methoxyfenozide‐treated male moths appear to be just as sensitive to various doses of synthetic codlemone as the antennae from the control and surfactant‐treated moths, but they appeared to be less sensitive to the pheromone component 12OH (collected from female effluvia) than the control male antennae. Mean male EAG responses to the pheromone components E8,E10‐12Al and codlemone collected from methoxyfenozide‐treated females were significantly less than the responses towards those two pheromone components collected from the control and surfactant‐treated females. Female moth exposure to methoxyfenozide did not negatively impact the sensitivity of female antennae to the plant volatile pear ester, but it did towards the apple volatile butyl hexanoate. Data from this study show that adult C. pomonella exposure to methoxyfenozide‐treated surfaces does appear to negatively impact, in a minor way, the (i) olfactory sensitivity (or detection) of male antennae towards some components of the female sex pheromone, (ii) the female antennal sensitivity towards a key apple volatile and (iii) the attractiveness of female pheromone effluvia.     

Chromosomal aberrations and sister-chromatid exchanges induced by N-nitroso-2-acetylaminofluorene and their modifications by arsenite and selenite in Chinese hamster ovary cells.

The frequencies of chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in Chinese hamster cells were significantly increased by the direct-acting mutagen N-nitroso-2-acetylaminofluorene (N-NO-AAF) at the concentration of 0.1 mM. N-NO-AAF was prepared by nitrosation of the protohepatocarcinogen 2-acetylaminofluorene. The induced CA, which included chromatid breaks, chromatid exchanges, chromosome breaks, and chromosome ring formation were significantly potentiated by the presence of sodium arsenite (10 microM), but not by hydroxyurea (20 mM) or cytosine arabinoside (25 microM). On the other hand, the clastogenic effect of N-NO-AAF was effectively inhibited by sodium selenite (100 microM). Arsenite (10 microM) was shown to be moderately active in CA induction which was partially blocked by the presence of selenite (10 nM). N-Nitroso compounds such as N-nitroso-N-methylurea, N-nitroso-N-ethylurea and N-methyl-N'-nitro-N-nitrosoguanidine were equally or more active in the induction of CA and SCE in CHO cells when compared with N-NO-AAF. The cell cycle was significantly delayed by the intervention of N-NO-AAF.     

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.