Engin Bermek

Mechanisms in polypeptide chain elongation on ribosomes.

Publisher Summary This chapter discusses that protein synthesis is a complex process involving multiple steps and requiring numerous cellular components. It reviews that the role of GTP in protein synthesis is the subject of numerous studies and speculations. It discusses the structure of the E. coli ribosome that provides insight into the ribosomal mechanisms at the molecular level. The translocation step as a central event of the ribosomal elongation process still remains as obscure as ever. Nonenzymic polypeptide synthesis may yield a suitable and simple assay system for investigating the interactions among tRNA, mRNA, and ribosomal components during translocation. The mechanism of elongation in eukaryotes is, in many ways, similar to the one in prokaryotes. Thus far, only EF-1 has proved to differ from the corresponding factor EF-T. The chapter also provides an overview on the eukaryotic elongation mechanism, its structure and mechanism of action of EF-1.

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.

New amphiphilic immunogens by poly(N-Isopropylacrylamide)-modified bovine serum albumin

Abstract Using poly (N-isopropylacrylamide) co-oligomer with N, N-dimethylacrylamide (DMAAm) (IDc) we prepared polymer-protein conjugates of bovine serum albumin (IDc-BSA). The conjugate underwent reversible hydration-dehydration changes in response to temperature changes and revealed phase separation at body temperature. Mice were immunized by the intravenous, intramuscular or intradermal routes with the IDc-BSA. The intravenous route of single immunization without adjuvants evoked increased primary and secondary specific immune responses to IDc-BSA, whereas the intramuscular and intradermal did not elicit higher antibody production. The intradermal administration of BSA and IDc-BSA together with IFA both gave rise to high immunological activity. It is suggested that the Hydrophobic chain aggregation site of the IDc-BSA conjugate at body temperature would increase the adsorptive capacity of BSA on the immunocompetent cells. The efficiency of such “forced” interactions of conjugate aggregates (high epitope density) are high enough for the immune response.

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

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.

Cloning and expression of the penicillin acylase gene (pac) from E. coli ATCC 11105

Abstract 1 The penicillin acylase gene ( pac ) from E. coli ATCC 11105 genomic DNA was cloned into pUC19 vector using conventional techniques. The E. coli strain JM109 transformed by this construct was shown to possess high plasmid stability. PAA upon initial addition to the cultivation medium inhibited cell growth as well as PA production in the recombinant strain. A repressor effect of glucose on PA production was also observed, although the construct did not contain the whole regulatory region of the pac gene. Regulation mechanisms of the pac gene are discussed in the light of previous data and the observed effects of PAA and glucose on penicillin acylase production by the recombinant strain. The penicillin acylase which was constitutively expressed by the recombinant strain was purified and optimal temperature and pH values were found to be 60°C and 8.5, respectively.

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.

beta-Thalassemia intermedia homozygous for normal hemoglobin A2 beta-thalassemia. Study in four families.

Four homozygotes for beta-thalassemia with normal hemoglobins A2 and F were studied. The absence or scarcity of transfusion requirement and comparatively low hemoglobin F content were the most important findings. Both parents of 3 patients showed the findings of beta-thalassemia with normal hemoglobins A2 and F. Biosynthetic studies in 2 patients and their both parents showed moderate or mild beta-chain deficiency. The possible reason for this comparatively mild course of a beta-thalassemia syndrome lies in a mild deficit in beta-chain production.

Hemin‐dependent induction and internalization of CD38 in K562 cells

The cell surface antigen, CD38, is a bifunctional ecto‐enzyme, which is predominantly expressed on hematopoietic cells during differentiation. In the present study, it is shown that hemin treatment of K562 cells gives rise to induction of enzymatic activities inherent to CD38. GDP‐ribosyl cyclase activity, an indicator of CD38, increased initially in response to hemin in a time‐dependent manner, reached a maximum level on the 5th day and, thereafter, declined sharply to the initial level. The increase in NAD+ glycohydrolase and ADP‐ribose uptake activities followed a similar time course. However, the decline in the latter activities after the 5th day of induction appeared to be rather slow in contrast to GDP‐ribosyl cyclase activity. The time course of these changes was well correlated with the FACScan findings obtained by use of anti‐CD38 monoclonal antibody. SDS–PAGE and Western blot analyses by use of the monoclonal antibody OKT10 revealed a transient hemin‐dependent appearence of a 43 kDa membrane protein with maximum signal intensity on the first 4 days of incubation. There was subsequently a gradual decrease on the 5th day, concomitant with a reciprocal increase in activity of the internalized protein fraction. The results together indicated that hemin‐induced expression of CD38 was followed by its down‐regulation. J. Cell. Biochem. 90: 379–386, 2003.