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

Oxidizing agents (e.g., H O ) cause structural and functional disruptions of molecules by affecting lipids, proteins, and nucleic acids.

K562 cells display different vulnerability to H₂O₂ induced oxidative stress in differing cell cycle phases.

Oxidative stress can be defined as the increase of oxidizing agents like reactive oxygen and nitrogen species, or the imbalance between the antioxidative defense mechanism and oxidants. Cell cycle checkpoint response can be defined as the arrest of the cell cycle functioning after damaging chemical exposure. This temporary arrest may be a period of time given to the cells to repair the DNA damage before entering the cycle again and completing mitosis.

Characterization of Lin⁻ALDH (bright) population using Ehrlich ascites tumor cells in mice.

Cancer stem cells (CSCs)/tumor initiating cells have been shown to exist in recent studies; however, it is challenging to isolate these cells. The latest evidence suggests that elevated aldehyde dehydrogenase (ALDH) activity is a hallmark of CSCs. In this study, mice implanted with Ehrlich ascites tumor (EAT) cells were used to isolate cancer stem cells.

Clinical significance of serum ADP-ribosylation and NAD glycohydrolase activity in patients with colorectal cancer.

The objective of this study was to evaluate the clinical significance of serum ADP-ribosylation and NAD glycohydrolase activity in patients with colorectal cancer (CRC). A total of 108 patients with CRC who underwent curative surgery and 20 healthy volunteers were enrolled in this study. ADP-ribosylation and NAD glycohydrolase activity levels were determined.

Purification of NAD glycohydrolase from human serum.

In the present study, NAD glycohydrolase was purified from serum samples collected from healthy individuals using ammonium sulfate fractionation, Affi-Gel blue (Cibacron Blue F3GA) affinity chromatography, Sephadex G-100 column chromatography and isoelectric focusing. The final step was followed by a second Sephadex G-100 column chromatography assay in order to remove the ampholytes from the isoelectric focusing step. In terms of enhancement of specific activity, the NAD glycohydrolase protein was purified ∼480-fold, with a yield of 1% compared with the initial serum fraction.

CD38 expression as response of hematopoietic system to cancer.

Erythrocyte and lymphocyte NAD(+) glycohydrolase levels were previously found to be elevated in cancer patients. These results were confirmed in an animal model. The administration of live Ehrlich ascites tumor cells to BALB/c mice led to increases in erythrocyte and lymphocyte NAD(+) glycohydrolase, along with tumor development.

Isolation of hematopoietic stem cells and the effect of CD38 expression during the early erythroid progenitor cell development process.

The aim of this study was to investigate changes in primitive hematopoietic cells through CD38 expression, identify the stage at which erythrocyte differentiation CD38 gains activity and the effects of serum factors on this expression by establishing a hematopoietic stem cell system in the erythroid development process. Using an immunomagnetic labeling and separation technique, CD34(+) cells were selected from cord blood. The CD34(+) cells were cultured in a 2 mM L-glutamine-enriched medium containing erythropoietin (Epo), penicillin-streptomycin and stem cell factor (SCF), and were incubated in 5% CO(2) at 37°C.

The cytotoxic effect of diphtheria toxin on the actin cytoskeleton.

Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin.

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.

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.

Erythrocyte CD38 as a prognostic marker in cancer.

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.

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.

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions.

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings.

NAD glycohydrolase activities and ADP-ribose uptake in erythrocytes from normal subjects and cancer patients.

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue.

Effect of oxidative stress on in vivo ADP-ribosylation of eukaryotic elongation factor 2.

Different lines of evidence indicate that eukaryotic elongation factor 2 (eEF2) can be ADP-ribosylated endogenously. The physiological significance of this reaction has, however, remained unclarified. In order to address this issue we investigated the in vivo ADP-ribosylation of eEF2 and the effect of oxidative stress thereon.

Actin--an inhibitor of eukaryotic elongation factor activities.

An inhibitor of diphtheria toxin- and endogenous transferase-dependent ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) has been found in the cytoplasmic fraction from rat liver. We provide evidence that this cytoplasmic inhibitor corresponds to actin, which gives rise also to inhibition of polyphenylalanine (polyPhe) synthesis. Both globular monomeric (G-actin) and filamentous (F-actin) forms of actin appear to be inhibitory on the action of elongation factors 1 and 2 (eEF1 and eEF2) in polyPhe synthesis with the inhibitory effect of G-actin proving to be stronger.