Yoshihiro Ojima

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation.

Metabolic alternations in SOD-deficient Escherichia coli cells when cultivated under oxidative stress from photoexcited titanium dioxide

Superoxide dismutase (SOD)-deficient Escherichia coli was cultivated under the oxidative stress generated by photoexcited titanium dioxide. These cells showed higher growth rate and glucose consumption rate with accelerated accumulation of acetic acid in the medium, compared to the cells cultivated under the normal condition without the stress. Under the stress condition, the activity of acetate kinase and mRNA expressions of the enzymes for acetic acid production (pta and ackA) were approximately doubled, while the activity of citrate synthase and mRNA expressions of the enzymes in TCA cycle (gltA, acnA, icd, sucA, sucC, sdhA, fumA and mdh) were repressed by about half, as compared with those under the normal condition. These results suggest that the stress-suffering cells switch the metabolic pathway into a “suppressed aerobiosis”, possibly for lowering the generation of reactive oxygen species.

Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles.

ABSTRACT Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.

Motility behavior of rpoS-deficient Escherichia coli analyzed by individual cell tracking.

Motility is one of the most extensively studied cellular events conducted by bacteria, including Escherichia coli. A motility agar plate assay showed that deletion of the rpoS gene enhanced the apparent motility of the E. coli BW25113 strain, which inherently had negligible motility compared to wild-type E. coli strains, such as MG1655, with no effect on cell growth. This enhancement of motility was accompanied by drastic up-regulation of genes involved in the formation and rotation of flagella. Furthermore, an individual cell motility assay showed that the population of ΔrpoS cells had bimodal motility character, and that a minority of this population exhibited a much higher motility rate. These results support a view that a minority population contributes to increasing in apparent motility of the whole population of ΔrpoS cells.

Promoted megakaryocytic differentiation of K562 cells through oxidative stress caused by near ultraviolet irradiation

Reactive oxygen species (ROS) have been proven to be important activators for various cellular activities, including cell differentiation. Several reports showed the necessity of ROS during cell differentiation of the megakaryocytic (MK) lineage. In this study, we employed near ultraviolet (near-UV) irradiation to generate endogenous oxidative stress in an MK differentiation process of K562 cells with phorbol 12-myristate 13-acetate (PMA) induction. A significant increase in the intracellular ROS level was detected on day 1 after near-UV irradiation. In the initial stage of differentiation, a shifted fraction of G1 and G2 phase cells was obtained using near-UV irradiation, giving an increased percentage of G2 phase cells (up from 31.1 to 68.7%). The near-UV irradiation-induced upregulation of the p21 gene, which is a cell cycle inhibitor, suggested that the G2 phase cells were prevented from undergoing cell division. It was found that the percentage of high ploidy (8N and 16N) cells was enhanced significantly at the later stage of the K562 cell culture with near-UV irradiation. Moreover, time-lapse analysis showed that near-UV irradiation encouraged the expression of CD41, a specific surface marker of megakaryocytes. This is the first report that the elevated oxidative stress through the near-UV irradiation promoted the MK differentiation of PMA-induced K562 cells.

Alternation in colonization behaviors of Escherichia coli cells with rpoS or yggE deficiency on solid surfaces

Colonization on a solid surface is influenced by the cell surface appendages such as flagella and curli, of which expressions are regulated by rpoS gene encoding a sigma factor. In this study, we investigated the effect of rpoS or yggE (a rpoS‐related and stress‐responsive gene) deficiency on the colonization of Escherichia coli BW25113. Under a static condition, the deletion of rpoS or yggE induced 3.9‐ and 3.7‐fold higher colonization as compared to wild‐type cells, respectively, on the solid surfaces. However, under a liquid flow condition, only ΔyggE cells maintained the stable colonization on the surface, and the values of cell layer thickness and cell coverage on the surface were 17 and 9.2 times as high as those of wild‐type cells, respectively. Gene expression analyses revealed that the deletion of rpoS or yggE positively impacted the expressions of genes involved in flagellum formation. On the other hand, curli assembly was severely prohibited by the rpoS deficiency. Here, we proposed that the plentiful flagella on the ΔrpoS and ΔyggE cell surfaces facilitated mainly the colonization under the static condition. Meanwhile, curli existing on the ΔyggE cell surface played an important role in keeping stable cell attachment and developing attached colonies under the flow stress condition. Biotechnol. Bioeng. 2013; 110: 1050–1056.

Exopolysaccharide assay in Escherichia coli microcolonies using a cleavable fusion protein of GFP-labeled carbohydrate-binding module

A fused protein composed of a carbohydrate-binding module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative evaluation of the EPShs.

An aerobic formate-utilizing bacterium, Cupriavidus sp., isolated from activated sludge of wastewater treatment

An aerobic formate-assimilating bacterium, denoted as strain FAB, was newly isolated from activated sludge of wastewater treatment. Phylogenetic analysis based on 16S rDNA sequence assigned the isolate to the genus Cupriavidus. Scanning electron micrography revealed that this bacterium has a coccal morphology, and from some physiological assays, the bacterium was characterized to be Gram-negative, nitrate-reduction-positive and catalase-positive. In addition to formate, strain FAB was able to utilize fructose, acetate or pyruvate as a preferred carbon source. Compared with a close relative, Cupriavidus necator, our isolate exhibited a greater growth rate on formate under an aerobic condition.

The rpoS deficiency suppresses acetate accumulation in glucose-enriched culture of Escherichia coli under an aerobic condition

The role of Escherichia coli rpoS on the central carbon metabolism was investigated through analyzing the deficiency of this regulon gene under aerobic and glucoseenriched culture conditions. The experimental results showed that while the wild type cells exhibited an overflow metabolism effect, the rpoS-deleting mutation alleviated this effect with the significant suppression of acetate accumulation under a high glucose condition. This gene deletion also induced the twofold upregulation of gltA and one-tenth downregulation of poxB, respectively. The overflow metabolism effect was confirmed to be recovered by re-introducing rpoS gene into the mutant. These results demonstrated rpoS changed the central carbon metabolism toward acetate overflow through dehydrogenation of pyruvate and reduction of TCA cycle activity.

Evaluation of photo-induced cellular damage using photoautotrophic cultures of puk-bung hairy roots as a sensing tool

In photoautotrophic cultures of pak-bung hairy roots, strong light irradiation (22 W/m(2)) increased the content of reactive oxygen species (ROS) in the cells, resulting in chlorophyll (Chl) degradation and DNA injury. The Chl degradation rate, R(D), can be used as a parameter to measure the cellular damage caused by photo-induced stress. The presence of the antioxidants ascorbic acid, chlorogenic acid or catechin reduced the R(D), while lowering the content of ROS and moderating the DNA injury.