Rüstem Nurten

Interactions of eukaryotic elongation factor 2 with actin: A possible link between protein synthetic machinery and cytoskeleton

Eukaryotic elongation factor 2 (EF‐2) was shown to bind to F‐actin as assayed by co‐sedimentation. In the presence of guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) binding was increased fourfold. At saturation level a molar ratio of about 0. 12 EF‐2 per F‐actin (subunit) was observed. Our results suggest a single type of binding site with an apparent dissociation constant of 0.85 μM. The stoichiometry was independent of the filament length, and ADP‐ribosylation had no effect on the binding. Experimental data indicated the involvement of SH‐groups of both EF‐2 and actin in the binding. The interaction EF‐2 with F‐actin appeared to be inhibited competitively by EF‐1α and non‐competitively by G‐actin.

Erythrocyte CD38 as a prognostic marker in cancer

Abstract Background: Surface antigen CD38 which is a multifunctional protein with enzymatic and receptorial properties is involved in many processes of cell proliferation and activation. It is widely expressed within the hematopoetic system, and its expression is stimulated by proinflammatory cytokines. CD38-associated enzymatic activities in erythrocytes from cancer patients were investigated in this context. Methods: Erythrocyte NAD glycohydrolase and ADP-ribosyl cyclase activities in normal individuals and cancer patients were compared and correlation of these activities to CEA values and anemia were determined. Changes in CD38-expression were followed by SDS-PAGE and Western blot analysis of erythrocyte membrane proteins. Results: Erythrocyte NAD glycohydrolase and ADP-ribosyl cyclase activities were significantly increased in cancer, in parallel to enhancement of CD38 expression and in correlation with CEA values and anemia. Conclusions: An increased expression of CD38 which may be due to action of proinflammatory cytokines produced in tumor–host reactions appears to account for the elevations in erythrocyte CD38-associated enzyme activities in cancer patients. The changes in these enzyme activities may provide a prognostic outlook in view of their apparently close correlation to tumor progressions.

Scopolamine-induced convulsions in fasted mice after food intake: determination of blood glucose levels, [3H]glutamate binding kinetics and antidopaminergic drug effects

The present study was performed to evaluate the role(s) of hypoglycemia, changes in [(3)H]glutamate binding kinetics and dopaminergic activity in the occurrence of scopolamine-induced convulsions in fasted mice after food intake. Plasma glucose levels and density (B(max)) and affinity (K(d)) of [(3)H]glutamate binding sites in whole brain synaptic membranes were determined in animals fed ad lib or fasted for 48 h and treated intraperitoneally (i.p.) with 3 mg/kg scopolamine or saline and allowed to eat for 5 min. Fasting for 48 h decreased plasma glucose levels. After refeeding, plasma glucose concentrations increased in saline treated animals, but remained unchanged in scopolamine treated animals which consumed less food. Fasting for 48 h also produced significant changes in the kinetics of [(3)H]glutamate binding. The B(max) and K(d) of the binding sites decreased in fasted animals. These changes were partially antagonized by scopolamine treatment and food intake. For the evaluation of the contribution of dopaminergic activity, another group of mice fasted for 48 h and pretreated (i.p.) with saline or dopamine antagonists, 2 mg/kg chlorpromazine or 2 or 4 mg/kg haloperidol, were treated 10 min later with either saline or 3 mg/kg scopolamine. Then 20 min later, they were allowed to eat ad lib and were observed for 30 min for the incidence and onset of clonic convulsions. Pretreatment of both 2 mg/kg chlorpromazine and 4 mg/kg haloperidol markedly suppressed the convulsions. These results indicate that the decrease in the [(3)H]glutamate binding induced by fasting, its antagonism by scopolamine treatment and food intake, and the dopaminergic hyperactivity may be possible factors contributing to the occurrence of convulsions.

Interaction of diphtheria toxin (fragment A) with actin

It was shown by gel filtration and viscosity measurements that N‐terminal fragment (FA) of diphtheria toxin (DT) can interact with both G‐ and F‐actin (filamentous actin). Elution profiles on Sephadex G‐100 indicated the formation of a binary complex of fragment A (FA) with globular actin monomer (G‐actin), which was inhibited by gelsolin. Deoxyribonuclease I (DNase I) in turn appeared to interact with this complex. Tritiated FA was found to bind to F‐actin stoichiometrically. This binding was inhibited again by gelsolin and G‐actin, but not by DNase I. The binding of FA inhibited polymerization of G‐actin and induced a time‐dependent breakdown of F‐actin under polymerization conditions. Inhibition of its ADP‐ribosyltransferase activity did not have any effect on the interactions of FA with actin. FA interacted with actin also in the cell. After treatment of human umbilical vein endothelial cells (HUVEC) with biotin‐labeled DT, Western blot analysis revealed predominantly the presence of actin in affinity‐isolated complexes of the labeled FA. Similarly, FA was found in immunoaffinity‐isolated complexes of actin. Copyright

Combined effects of epirubicin and tamoxifen on the cell-cycle phases in estrogen-receptor-negative Ehrlich ascites tumor cells

Laboratory and clinical data suggest some interactions between cytotoxic agents and tamoxifen. The mechanisms of these interactions differ in estrogen-receptor-negative cell lines. The ability of tamoxifen to modify the effects of epirubicin on the cell-cycle phases of estrogen-receptor-negative Ehrlichs carcinoma ascitic cells (EATC) was studied in mice. The results showed that combination of tamoxifen with epirubicin decreased the thymidine labelling index more effectively than did either drug alone. Adding tamoxifen to epirubicin treatment induced both an early S-phase and G2-M-phase arrest and a later G0-G1-phase arrest in EATC. An increase of S0 cells in the quiescent fraction could play a role in these changes, and some of these quiescent cells may not be viable, causing them to die later. In conclusion, the data suggest that continuous exposure to tamoxifen might modify the effects of epirubicin via cell-cycle perturbations.

Functional groups of elongation factor 2 involved in interactions with guanosine nucleotides and ribosomes.

Treatment of rat liver EF‐2 with N‐ethylmaleimide (MalNEt) did not affect the direct interactions of the factor with guanine nulceotides or with ribosomes, but inhibited the binding of guanosine 5′‐(β,γ‐methylene)triphosphate (GuoPP(CH2)P) to the EF‐2‐ribosome complex. The amino group reactive reagent 2,4,6‐trinitrobenzenesulfonate (TNBS), however, inhibited specifically the direct interactions of EF‐2 with guanine nucleotides, but not the binding of GuoPP(CH2)P to the EF‐2‐ribosome complex. The different sensitivities of EF‐2 to MalNEt and to TNBS suggested that the binding sites involved in the binary vs. ternary complex might correspond to different conformational states or might even be distinct physical entities.

β-carotene treatment alters the cellular death process in oxidative stress-induced K562 cells†

Oxidizing agents (e.g., H2O2) cause structural and functional disruptions of molecules by affecting lipids, proteins, and nucleic acids. As a result, cellular mechanisms related to disrupted macro molecules are affected and cell death is induced. Oxidative damage can be prevented at a certain point by antioxidants or the damage can be reversed. In this work, we studied the cellular response against oxidative stress induced by H2O2 and antioxidant–oxidant (β‐carotene–H2O2) interactions in terms of time, concentration, and treatment method (pre‐, co‐, and post) in K562 cells. We showed that co‐ or post‐treatment with β‐carotene did not protect cells from the damage of oxidative stress furthermore co‐ and post‐β‐carotene‐treated oxidative stress induced cells showed similar results with only H2O2 treated cells. However, β‐carotene pre‐treatment prevented oxidative damage induced by H2O2 at concentrations lower than 1,000 μM compared with only H2O2‐treated and co‐ and post‐β‐carotene‐treated oxidative stress‐induced cells in terms of studied cellular parameters (mitochondrial membrane potential [Δψm], cell cycle and apoptosis). Prevention effect of β‐carotene pre‐treatment was lost at concentrations higher than 1,000 μM H2O2 (2–10 mM). These findings suggest that β‐carotene pre‐treatment alters the effects of oxidative damage induced by H2O2 and cell death processes in K562 cells.

On diphtheria toxin fragment A release into the cytosol--cytochalasin D effect and involvement of actin filaments and eukaryotic elongation factor 2.

Diphtheria toxin has been well characterized in terms of its receptor binding and receptor mediated endocytosis. However, the precise mechanism of the cytosolic release of diphtheria toxin fragment A from early endosomes is still unclear. Various reports differ regarding the requirement for cytosolic factors in this process. Here, we present data indicating that the distribution of actin filaments due to cytochalasin D action enhances the retention of diphtheria toxin in early endosomes. Treating cells with cytochalasin D reduces the cytosolic fragment A activity and leads to changes in the intracellular distribution and size of early endosomes with toxin cargo. F-actin and eukaryotic elongation factor 2 can promote fragment A release from toxin-loaded early endosomes in an in vitro translocation system. Moreover, these proteins bind to toxin-loaded early endosomes in vitro and promote each others binding. They are thus thought to be involved in the cytosolic release of fragment A. Finally, ADP-ribosylation of eukaryotic elongation factor 2 is shown to inhibit fragment A release and, via a feed-back mechanism, to account for the minute amounts of fragment A normally found in the cytosol.

ADP-ribosylation of serum proteins : Elevated levels in neoplastic cases due to altered NAD/ADP-ribose metabolism

ADP-ribosylation of human serum proteins was studied in various groups of disorders. In most of these groups, the extent of ADP-ribosylation did not show a divergence from the group of normal controls. Neoplastic diseases revealed, however, a unique group, with more than fivefold increases in ADP-ribosylation levels over the other groups. Blood samples with high levels of ADP-ribosylation revealed, in general, increased serum NAD glycohydrolase activities and low levels of serum NAD.

ADP-ribosylation of serum proteins: evaluation as a potential tumor marker.

Serum samples from cancer patients revealed elevated levels of in vitro ADP-ribosylation through non-enzymic binding of ADP-ribose to free acceptor sites on serum proteins. Low concentrations of serum ADP-ribose caused by high NAD glycohydrolase activity together with elevated rates of ADP-ribose transport into erythrocytes appeared to account for under-saturation of the acceptor sites on serum proteins. ADP-ribosylation of serum proteins was assessed as an indicator of cancer disease, and an attempt was made to determine the correlation of ADP-ribosylation levels with carcinoembryonic antigen (CEA) values. Based on positive test results for all tumor patients and negative test results for all healthy controls, sensitivity and specificity of ADP-ribosylation as a tumor indicator were estimated as 67% and 95%, respectively. A close correlation appeared to exist with CEA (r = 0.67; P < 0.001). Similarly, the changes in the levels of ADP-ribosylation correlated with the changes in the levels of CEA during the clinical course (r = 0.58; P < 0.05).