The sulphation inhibitor sodium selenate arrests the growth of Dictyostelium discoideum

Abstract Sulphate incorporation into glycopeptides appears to be a key event in the development of a number of organisms. An inhibitor of sulphation, sodium selenate, has been used in this study to examine the possibility that sulphation has a comparable role in the development of Dictyostelium discoideum . At concentrations of 0.1 mM and 1.0 mM, exogenously supplied selenate reversibly arrested the growth of bacterially grown amoebae of D. discoideum . In contrast, the effect of selenate on development was minimal. In the presence of 1.0 mM selenate, aggregation and tip formation were delayed 2–3 h and aggregates were slightly smaller; exogenous 0.1 mM selenate had no visible effect on development. However, the possibility that starved amoebae are impermeable to selenate was not excluded. The vegetative growth and development of an axenic strain in the presence of selenate closely resembled that of the bacterially grown strain. Since an inhibitory effect of 1.0 mM selenate on [³⁵S]sulphate incorporation into acetone precipitable protein was also demonstrated, these results suggest that sulphation is necessary for the growth of D. discoideum .     

Reduction of Selenate to Selenide by Sulfate-Respiring Bacteria: Experiments with Cell Suspensions and Estuarine Sediments

Washed cell suspensions of Desulfovibrio desulfuricans subsp. aestuarii were capable of reducing nanomolar levels of selenate to selenide as well as sulfate to sulfide. Reduction of these species was inhibited by 1 mM selenate or tungstate. The addition of 1 mM sulfate decreased the reduction of selenate and enhanced the reduction of sulfate. Increasing concentrations of sulfate inhibited rates of selenate reduction but enhanced sulfate reduction rates. Cell suspensions kept in 1 mM selenate were incapable of reducing either selenate or sulfate when the selenate/sulfate ratio was ≥0.02, indicating that irreversible inhibition occurs at high selenate concentrations. Anoxic estuarine sediments having an active flora of sulfate-respiring bacteria were capable of a small amount of selenate reduction when ambient sulfate concentrations were low (<4 mM). These results indicate that sulfate is an inhibitor of the reduction of trace quantities of selenate. Therefore, direct reduction of traces of selenate to selenide by sulfate-respiring bacteria in natural environments is constrained by the ambient concentration of sulfate ions. The significance of this observation with regard to the role sediments play in sequestering selenium is discussed.     

Effect of byproduct,nitrogen fertilizer,and zeolite on phosphate rock dissolution and extractable phosphorus in acid soil

The relative plant availability of selenate versus selenite depends on the concentrations of competing ions, specifically sulfate and phosphate, respectively. In solution culture, the concentration of phosphate is typically 100- to 1000-fold greater than in soil solution, an artifact that could lead to underestimation of the phytoavailability of selenite. A nutrient solution study was conducted to compare the availability of selenite and selenate to perennial ryegrass (Lolium perenne L. cv. Evening Shade) and strawberry clover (Trifolium fragiferrum L. cv. O'Conner) at basal concentrations of SO₄ (0.5 mM) and PO₄ (2 μM) similar to those found in soil solution. Concentrations up to 5 mM SO₄ and 200 μM PO₄ allowed quantitative comparison of the efficacy of the competing ions. In both species, selenite was more phytotoxic than selenate, especially for shoot growth. Selenate was less toxic, and tended to preferentially inhibit root growth. Translocation percentages were much higher with selenate (≥84%) than with selenite (≤47%). A 10-fold increase in sulfate decreased uptake from selenate by >90% in both species. In ryegrass, 10-fold increases in phosphate caused 30% to 50% decreases in Se accumulation from selenite, but in clover such decreases only occurred in the roots. Sulfate-selenate antagonisms were thus stronger than phosphate-selenite antagonisms. Nonetheless, conventional nutrient solutions with millimolar phosphate will significantly underestimate Se availability from selenite, and moderate levels of sulfate salinity can inhibit selenate uptake sufficiently to reverse the apparent relative availability of the two forms of Se. This revised version was published online in June 2006 with corrections to the Cover Date.     

Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with alpha and beta subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (alphabetagamma) complex with an apparent molecular mass of approximately 600 kDa. The individual subunit sizes are approximately 100 kDa (alpha), approximately 55 kDa (beta), and approximately 36 kDa (gamma), with a predicted overall subunit composition of alpha3beta3gamma3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be approximately 2 mM, with an observed Vmax of 500 nmol SeO4(2-) min(-1) mg(-1) (kcat, approximately 5.0 s(-1)). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

The insulin-like effects of selenate in rat adipocytes

Selenate was found to have several insulin-like effects in rat adipocytes: stimulation of glucose transport activity by translocation of two types of glucose transporters from intracellular sites to the plasma membrane, stimulation of cAMP phosphodiesterase activity, and stimulation of ribosomal S6 protein phosphorylation. Furthermore, in intact cells addition of 1 mM selenate stimulated tyrosyl phosphorylation of 210-, 170-, 120-, 95-, 70-, and 60-kDa proteins but failed to stimulate insulin receptor kinase activity, suggesting that selenate stimulated other tyrosine kinase. In the presence of insulin, selenate enhances insulin receptor kinase activity and phosphorylations of insulin-stimulated tyrosyl phosphoproteins. These results may provide clues for the elucidation of the role of selenium in animals and the mechanism of insulin action.     

Characterization of the reduction of selenate and tellurite by nitrate reductases.

Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V(max) and K(m) were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 micromol x min(-1) x mg(-1) and 0.12 mM were obtained for V(max) and K(m), respectively, whereas the V(max) values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.     

Effect of selenium compounds and thiols on human mammary tumor cells

The effect on cell viability and growth rate of sodium selenite, selenocystine, sodium selenate, and selenomethionine at selenium concentrations of 6.25 and 12.5 uM was studied in vitro on cells of the human mammary tumor cell line HTB123/DU4475. Selenite and selenocystine affected both cell viability and growth rate of the tumor cells at these selenium concentrations. Selenite and selenocystine decreased intracellular glutathione concentrations, but did not affect tumor cell glutathione peroxidase activity. After six days of exposure to either selenate or selenomethionine, the viability of tumor cells remained stable, but cell growth, as measured by numbers of cells, was retarded. Neither selenate nor selenomethionine produced changes in concentrations of intracellular glutathione. The toxic effect of selenite on tumor cells was enhanced by addition of 0.25 mM glutathione to the growth medium. Preincubation of the tumor cells with 62.5 uM buthionine sulfoximine decreased cellular glutathione to 15% of controls at 24 h and enhanced the toxicity of selenite toward the tumor cells. Glutathione, 2-mercaptoethanol, and L-cysteine were all toxic to the tumor cells in a dose-dependent manner.     

Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced (35)S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.     

Sulphate transport in human placental brush-border membrane vesicles: competitive inhibition by selenate

The effect of selenate on sulphate uptake by human placental brush-border membrane vesicles has been investigated. Selenate added to the incubation medium inhibits 1 mM sulphate uptake in a dose-dependent fashion with a Ki of approx. 2.5 mM. The inhibition by selenate is competitive, suggesting that selenate and sulphate share a common transporter (an anion exchange system) which may be of particular importance for the transport of such essential trace elements to the fetus.     

Removal of soluble selenium by a selenate-reducing bacterium Bacillus sp. SF-1

In order to develop a biological process for removal of selenium from industrial wastewater, Bacillus sp. strain SF-1 was isolated from selenium-contaminated sediment. The bacterium reduces selenate to selenite and subsequently to nontoxic insoluble elemental selenium using lactate as an electron donor and selenate as an electron acceptor in an anaerobic condition. Elemental selenium transformed from soluble selenium was deposited both inside and outside of the cells. Since the selenate reduction rate of the strain SF-1 was higher than the selenite reduction rate, selenite was transiently accumulated. In an experiment of the repeated soluble selenium reduction by strain SF-1, 0.5 mM of selenate was sequentially treatable with a cycle of one day. Thus, our sequential system for removal of soluble selenium is very useful.     

Adenosine 5'-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity

APR2 is the dominant APR (adenosine 5'-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species).     

The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana

To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.     

Impact of sulphur fertilisation on crop response to selenium fertilisation

UK wheat (Triticum aestivum L.) has a low selenium (Se) concentration and agronomic biofortification with Se is a proposed solution. A possible limitation is that UK wheat is routinely fertilised with sulphur (S), which may affect uptake of Se by the crop. The response of wheat to Se and S fertilisation and residual effects of Se were determined in field trials over 2 consecutive years. Selenium fertilisation at 20 g ha^?1 as sodium selenate increased grain Se by four to seven fold, up to 374 µg Se kg^?1. Sulphur fertilisation produced contrasting effects in 2 years; in year 1 when the crop was not deficient in S, grain Se concentration was significantly enhanced by S, whereas in year 2 when crop yield responded significantly to S fertilisation, grain Se concentration was decreased significantly in the S-fertilised plots. An incubation experiment showed that addition of sulphate enhanced the recovery of selenate added to soils, probably through a suppression of selenate transformation to other unavailable forms in soils. Our results demonstrate complex interactions between S and Se involving both soil and plant physiological processes; S can enhance Se availability in soil but inhibit selenate uptake by plants. Furthermore, no residual effect of Se fertiliser applied in year 1 was found on the following crop.     

Significant Improvement of Insulin Resistance of GK Rats by Treatment with Sodium Selenate

We studied the effect of sodium selenate on insulin resistance of Goto-Kakizaki (GK) rats. Rats were kept on standard laboratory chow with and without i.p. injections of sodium selenate (0.173 mg/kg body weight) for 14 days, and then subjected to the glucose clamp. The glucose clamp studies confirmed an improvement in insulin-stimulated glucose disposal in GK rats treated with sodium selenate, with respect to both insulin sensitivity and responsiveness. This amelioration of insulin resistance may be partly due to a direct effect of the sodium selenate on peripheral tissues. 2-Deoxyglucose uptake in sodium selenate-treated adipocytes was increased and the insulin findings suggest that sodium selenate increases not only insulin sensitivity but also insulin responsiveness. Sodium selenate also accelerated glucose incorporation into adipocytes of rats, suggesting that the action of sodium selenate is peripheral. Interestingly, sodium selenate at a high concentration (about 1 mmol/L) was more effective than insulin in enhancing glucose uptake. The present study suggested that sodium selenate treatment led to substantial improvement in peripheral insulin resistance.     

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

An interaction between selenate and a sulfate transporter in the lactating rat mammary gland.

The efficacy of selenate and other divalent anions to stimulate the efflux of radiolabeled sulfate from lactating rat mammary tissue slices has been examined. Both selenate and sulfate markedly increased the fractional release of sulfate via a system that is temperature-sensitive and sensitive to the anion-exchange inhibitor DIDS. The effect of selenate on sulfate efflux was saturable with an apparent affinity constant of approximately 0.27 mM. In addition, molybdate and thiosulfate were also found to increase sulfate efflux from the trans-aspect. It is concluded that sulfate and selenate share a pathway for transport in the lactating rat mammary gland.     

Overexpression of ATP Sulfurylase in Indian Mustard Leads to Increased Selenate Uptake, Reduction, and Tolerance

In earlier studies, the assimilation of selenate by plants appeared to be limited by its reduction, a step that is thought to be mediated by ATP sulfurylase. Here, the Arabidopsis APS1 gene, encoding a plastidic ATP sulfurylase, was constitutively overexpressed in Indian mustard (Brassica juncea). Compared with that in untransformed plants, the ATP sulfurylase activity was 2- to 2.5-fold higher in shoots and roots of transgenic seedlings, and 1.5- to 2-fold higher in shoots but not roots of selenate-supplied mature ATP-sulfurylase-overexpressing (APS) plants. The APS plants showed increased selenate reduction: x-ray absorption spectroscopy showed that root and shoot tissues of mature APS plants contained mostly organic Se (possibly selenomethionine), whereas wild-type plants accumulated selenate. The APS plants were not able to reduce selenate when shoots were removed immediately before selenate was supplied. In addition, Se accumulation in APS plants was 2- to 3-fold higher in shoots and 1.5-fold higher in roots compared with wild-type plants, and Se tolerance was higher in both seedlings and mature APS plants. These studies show that ATP sulfurylase not only mediates selenate reduction in plants, but is also rate limiting for selenate uptake and assimilation.     

Influence of alpha-tocopherol on prostacyclin synthesis by rat's arterial and myometrial tissues

Influence of alpha-tocopherol on PGI2 synthesis by rat arterial and myometrial tissues was investigated using a rat platelet antiaggregatory bioassay. Chronic administration of alpha-tocopherol to female rats (10 mg kg-1 day-1 s.c. for 14 days) significantly increased ex-vivo PGI2 synthesis by the arterial tissue from 12.7 +/- 0.3 (control, mean +/- s.e.m) to 17.2 +/- 0.4 ng PGI2 mg-1 wet tissue and by the myometrial tissue (in proestrus) from 1.1 +/- 0.07 (control) to 1.85 +/- 0.1 ng PGI2 mg-1 wet tissue (P less than 0.05, n = 6). alpha-tocopherol (5 mg kg-1 day-1 for 14 days) did not stimulate PGI2 to any significant level. Pretreatment of male rat arterial tissue with alpha-tocopherol (0.02, 0.1 or 0.2 mM) in vitro increased PGI2 synthesis in a dose-dependent manner. At a dose of 0.2 mM it increased PGI2 synthesis from 13.70 +/- 0.70 (control) to 22.6 +/- 1.4 ng PGI2 mg-1 wet tissue (P less than 0.1, n = 6). Pre-treatment of 14-day pregnant rat myometrium with alpha-tocopherol 0.2 and 0.4 mM significantly increased PGI2 synthesis from 1.2 +/- 0.06 (control) to 1.90 +/- 0.12 and 2.1 +/- 0.1 ng PGI2 mg-1 wet tissue, respectively (P less than 0.05, n = 6). The results indicate that the ability of alpha-tocopherol to stimulate PGI2 synthesis may partly contribute towards better understanding of the mechanisms that underly the protective effect of alpha-tocopherol against experimentally induced decreases in coronary flow and intravascular coagulations in some mammals. Furthermore adequate intake of alpha-tocopherol during pregnancy may enhance uterine blood flow and ensure adequate foetal nutrition.     

Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ~600 kDa. The individual subunit sizes are ~100 kDa (α), ~55 kDa (β), and ~36 kDa (γ), with a predicted overall subunit composition of α₃β₃γ₃. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent K_m for selenate was determined to be ~2 mM, with an observed V_max of 500 nmol SeO₄²⁻ min⁻¹ mg⁻¹ (k_cat, ~5.0 s⁻¹). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

db—cAMP对转化细胞钙调素基因表达与细胞骨架的影响

We have demonstrated that the distribution of microtubules (MT), microfilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c-fos enhanced in the transformed C3 H10 T1/2 cells. After treatment with 1 mM db-cAMP for 1 hr. and 2 hrs., there was an early and rapidly reduced in gene expression of calmodulin and c-fos respectively. After db-cAMP treatment for 4-5 days, the number of Capping cells of ConA binding decreased significantly and the cell surface microvilli decreased also. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed in G1 phase, we have found that the db-cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, microfilaments and fibronectin were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the inhibition of proliferation, alteration of phenotype and recovery of cytoskeleton in transformed cells after treatment with db-cAMP are related to the inhibition of gene expression of calmodulin.