The effects of leptin on F-actin remodelling in type 1 diabetes.

BACKGROUND: The aim of the current study is to investigate the effect of leptin on cytoskeleton structures in both in vivo and in vitro model of diabetes. MATERIALS AND METHODS: For in vivo studies, leptin in different doses (240, and 480 mg/kg) was injected to the diabetic rats after 1-week of streptozotocin (STZ, 55 mg/kg) treatment. Leptin levels were analysed in serum, liver, and pancreas samples. Hepatic and pancreatic F- and G-actin expressions were determined by Western blotting. For in vitro studies, hepatic and pancreatic primary cell lines were obtained from the control rats. To these cultures, STZ (15 and 30 mM), leptin (50, 60 and 100 ng/mL), and their combinations were applied for 1, 3, and 4 weeks. After the treatment period, F-actin was visualised by the Alexa-fluor fluorescent dye. RESULTS: Streptozotocin decreased the G-actin in both tissues in vivo. However, leptin caused a dose-dependent increase in G-actin levels while F-actin decreased in both tissues. Moreover, leptin caused the perimembranous condensation of actin filaments and amelioration of F-actin structures in vivo. A dose-dependent corruption of F-actin filament structures was observed in leptin-treated primary cells in vitro, while STZ also caused corruption of these filaments. Co-exposure of STZ and leptin caused the amelioration of F-actin filaments, while the peri- membranous condensation was also observed as was in vivo study. CONCLUSIONS: Leptin therapy could be a candidate for diabetes, but it should not be ruled out as being important the severity of diabetes and leptin doses.

The interaction between actin and FA fragment of diphtheria toxin.

Actin protein has many other cellular functions such as movement, chemotaxis, secretion and cytodiaresis. Besides, it have structural function. Actin is a motor protein that it has an important role in the movement process of toxin in the cell. It is known that F-actin gives carriage support during the endosomal process. Actin is found in globular (G) and filamentous (F) structure in the cell. The helix of actin occurs as a result of polymerisation of monomeric G-actin molecules through sequential rowing, is called F-actin (FA). Actin interacts with a great number of cellular proteins along with cell skeleton and plasma membrane. It is also known that some bacterial toxins have ADP-ribosylation affect on actin. Diphteria toxin is the part which has the FA enzymatic activity corresponding the N-terminal section of the toxin, which inhibits the protein synthesis by ADP-ribosylating the elongation factor 2 in the presence of NAD. FA, taken into the cell by endocytosis inhibits protein synthesis by ADP-ribosyltransferase activity and breaks the cytoskeleton. In the studies both in vitro and in vivo, actin with interaction FA of diphteria toxin has been yet to be fully elucidated. The aim of this study was to determine the three dimensional structures of actin with interaction FA of diphteria toxin by the amprical methods and in paralel with the computing technology, theoretical methods have gained significant importance. In our study, actin with interaction FA of diphteria toxin has been determined as the most possible interaction area with the theoretical method; analogy modelling. This area has been closed in the presence of polypeptides and FA-actin interactions have been tested with the gel filtration chromatography techniques. As a result of the findings, we found that 15 amino acid artificial peptides (DAMYETMAQACAGNR) corresponding to 201-215 amino acid residues of FA interacts with G-actin and closes this area. Secondly, in the model formed with the analogy modelling, it appears that the most possible interaction area is between FA (tyr204) and G-actin (gly48). Results obtained from both theoretical and experimental data support the idea that the interaction occurs in this area.

Endogenous ADP-ribosylation of eukaryotic elongation factor 2 and its 32 kDa tryptic fragment

Eukaryotic elongation factor 2 (eEF-2) can undergo ADP-ribosylation in the absence of diphtheria toxin. The binding of free ADP-ribose and endogenous transferase-dependent ADP-ribosylation were distinct reactions for eEF-2, as indicated by different findings. Incubation of eEF-2 tryptic fragment 32/33 kDa (32F) with NAD was ADP-ribosylated and gave rise to the covalent binding of ADP-ribose to eEF-2. 32F was revealed to be at the C-terminal by Edman degradation sequence analysis. In our study, the elution of 32F from SDS-PAGE was ADP-ribosylated both in the presence and absence of diphtheria toxin. These results suggest that endogenous ADP-ribosylation of 32F might be related to protein synthesis. This modification appears to be important for the cell function.

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.

The importance of the number of NMDA receptors in the development of supersensitivity or tolerance to and dependence on morphine.

Opiate or NMDA receptor antagonists given during and/or after the development of tolerance and dependence have been reported to prevent these developments. In the present study, MK801 (dizolcipine) and naltrexone (NX), two antagonists of NMDA and opiate receptors, respectively were used in rats to find any correlations between changes in NMDA receptor kinetics, and the intensity of tolerance and dependence. Thus, six different groups of rats were formed. The rats in the groups were given saline (S)+S, S+morphine (M), NX+S, NX+M, MK801+S and MK801+M, respectively, once per day for 8 days. On day 9, the rats from each group were divided into four subgroups. The rats of the first subgroup were subjected to the determination of tail-flick latency. The rats of the second subgroup were administered 1 mg kg-1 naloxone (NL) 2 h after administration of 3 mg kg-1M. The rats of the third subgroup were implanted with two M pellets and after 72 h they were challenged with NL. The remaining rats received drugs also on day 9 according to the previous administration paradigm. Two hours after the administrations, their brains were utilised for the determination of NMDA receptor kinetics, employing [3H]glutamate. The measurement of tail-flick latency showed the prevention by NX or MK801 of the development of tolerance to M. The rats, which were administered 3 mg kg-1M 2 h before 1 mg kg-1 NL injection, on day 9 showed that only NX given previously along with M attenuated the intensity of the development of M dependence. NX administered alone intensified the development of dependence on a single dose of M. The development of M dependence upon the M pellet implantation was intensified by the previous administration of NX or MK801 concomitantly with M. The administration of M or MK801 alone, or NX together with M, caused significant upregulation of NMDA receptors. NX alone, and MK801 given concurrently with M led to a significant downregulation. So, in light of the previous findings and the present experimental data it can be said that: (1) supersensitivity to opioids may be a downregulation of NMDA as well as an upregulation of the opioid receptor; (2) either upregulation or downregulation of NMDA receptors may facilitate subsequent development of opioid dependence; (3) tolerance to opioid may necessitate both upregulation of NMDA receptors and downregulation of opioid receptors; and (4) beneficial effects of opioid antagonists in the treatment of opiate dependence and CNS injuries may be strongly related to the down regulation of NMDA receptors.

On the mode of inhibition of eukaryotic protein synthesis by ADP-ribosylation of elongation factor 2.

The exchange of free guanine nucleotides with guanine nucleotides bound to elongation factor 2 (EF-2) and to the EF-2-ribosome complex, and the effect of ADP-ribosylation of the EF-2 thereon, were investigated by nitrocellulose filter assay. Under the experimental conditions, stoichiometric amounts of guanine nucleotides were bound, in particular, to ternary complexes of EF-2 with biphasic kinetics. The exchange kinetics were similarly biphasic in all cases. Ribosomes appeared to have variable effects on the exchange kinetics, depending on the type of nucleotide bound. Thus, in their presence, the rate and magnitude of the fast exchange of nucleotides revealed increasing values in the order GTP (GXP) > GTP gamma S > GDP. ADP-ribosylation had no inhibitory effect on the binding of guanine nucleotides to EF-2 or to the EF-2-ribosome complex but reduced significantly the fast exchange of GTP (GXP) and GTP gamma S bound to the EF-2-ribosome complex. The effect of ADP-ribosylation on the fast exchange of GDP in binary and ternary complexes was less pronounced. The mechanism of inhibition of protein synthesis by ADP-ribosylation of EF-2 is discussed in view of these data.

Interactions of elongation factor 2 with the cytoskeleton and interference with DNase I binding to actin.

Interactions of elongation factor 2 (EF-2) with G-actin and F-actin in vitro were investigated using viscosimetry, gel filtration and electron microscopy. Under depolymerization conditions, at a molar ratio of 0.5:1 (EF-2/F-actin subunit), F-actin is stabilised by EF-2 and filaments depolymerize about three times slower than control solutions containing only F-actin. Filament stability is improved also when EF-2 is included in the solution in the presence of DNase I. Electron micrographs and viscosity measurements indicate that EF-2 may support small bundles with a width of 2 or 3 filaments. It was established that EF-2 interacts with G-actin in vitro, and reduces G-actin inhibition of DNase I activity when it is present at a ratio of 1:1. Results are discussed in the context of possible functional significance of the interactions.

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.

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 Ehrlich's 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.

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).

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) (GTP gamma S) 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 microM. 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-1 alpha and non-competitively by G-actin.

ADP-ribosylation of human serum proteins promoted by endogenous NAD glycohydrolase activity.

Incubation of human serum samples with [adenine-14C]NAD resulted in a time- and dose-dependent incorporation of adenine moiety into CCI3COOH-precipitable material. Incorporated radioactivity was relatively resistant to neutral hydroxylamine, but was completely released by treatment with NaOH. An incorporation was observed also after preincubation of NAD with NAD glycohydrolase from pig brain. NAD glycohydrolase activity in serum samples was then shown spectroscopically in an assay coupled to alcohol oxidation. Thus, this reaction was implicated to be due to the binding of ADP-ribose, formed under the action of a soluble, endogenous NAD glycohydrolase activity, to serum proteins. Analysis by NaDodSO4/polyacrylamide gel electrophoresis (PAGE) and autoradiography indicated that a polypeptide of 97 kD, but also two further polypeptides of higher molecular weight and serum albumin, were labelled after incubation with radioactive NAD.

On the nature of cellular ADP-ribosyltransferase from rat liver specific for elongation factor 2.

A cellular ADP-ribosyltransferase, specific for elongation factor 2 (EF-2), is found in extracts from rat liver. Co-migrating with EF-2 throughout purification, this activity is, moreover, located in the protein bands corresponding to EF-2 after native or sodium dodecyl sulfate polyacrylamide gel electrophoresis. The observed activity is thus implicated to be an inherent property of EF-2. Preincubation of EF-2 with GuoPPCH2Pox inhibits endogenous, but not diphtheria toxin catalyzed ADP-ribosylation.

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 nucleotides or with ribosomes, but inhibited the binding of guanosine 5'-(beta, gamma-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.

Interactions of elongation factor 2 (EF-2) with guanine nucleotides and ribosomes. Binding of periodate-oxidized guanine nucleotides to EF-2.

Interactions of rat liver elongation factor 2 (EF-2) with guanine nucleotides and ribosomes were studied by equilibrium dialysis and sedimentation methods. GDP (Kd = 0.5 microM) or GDP-Mg2+ (Kd = 1.57 microM) displayed a higher affinity in the formation of a binary complex with EF-2 than GTP (Kd = 2.68 microM), GTP-Mg2+ (Kd = 2.77 microM), or guanosine 5'-[beta, gamma-methylene]triphosphate (GuoPP[CH2]P) (Kd = 24.0 microM). NaIO4-oxidized guanine nucleotides (oGDP) (Kd = 38 microM) and oxidized/reduced guanine nucleotides (orGDP) (Kd = 27 microM) had lower affinites to the binding site on EF-2 than those of GDP or GTP. However, the binding of oGDP, oGTP or oGuoPP[CH2]P to EF-2 resulted in the formation of a stable product which could be recovered by the nitrocellulose filter technique or by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In the presence of ribosomes and EF-2 the formation of a new binding site (or a different conformation of the binding site) with a higher affinity for GuoPP[CH2]P-Mg2+ (Kd = 0.26 microM) than fof GDP-Mg2+ (kd = 9.3 microM) became apparent. The presence of ribosomes thus appeared to favor the formation of a complex involving guanosine triphosphates. Adenosine diphosphate ribosylated EF-2 (ADP-Rib-EF-2) in its turn could bind to the ribosome with high affinity even without guanosine nucleotides (Kd = 0.18 microM). GuoPP[CH2]P increased to some extent the affinity of ADP-Rib-EF-2 for its ribosomal binding site (Kd = 0.05 microM).