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Indium tin oxide (ITO) nanoparticles triggered the release of IL-1β from macrophages, followed by the significant induction of epithelial-mesenchymal transition (EMT) in alveolar epithelial cells.

In this study, the cellular effects of lead (Pb) nanoparticles with a primary particle size of 80 nm were evaluated in two types of cell lines: human lung carcinoma A549 and macrophage-differentiated THP-1 cells (dTHP-1). The cellular responses induced by the Pb nanoparticles varied among the cell types. Exposure to Pb nanoparticles for 24 h at a concentration of 100 μg/ml induced interleukin-8 (IL-8) expression in dTHP-1 cells. Induction of IL-8 expression in A549 was lower than dTHP-1 cells. Pb nanoparticles also induced the gene expression of heme oxygenase-1 in dTHP-1 cells but not in A549 cells. Though cellular uptake of Pb nanoparticles was observed in both the cell types, the amount of internalized Pb particles was lower in A549 cells than that in dTHP-1 cells. Gene expression of metallothionein 2A was remarkably enhanced by Pb nanoparticle exposure in dTHP-1 cells. Compared with Pb nanoparticles, induction of cytokines caused by lead nitrate (Pb[NO3 ]2 ), a water-soluble Pb compound, was smaller. In conclusion, the present study revealed that Pb nanoparticles induced a stronger cellular response than Pb(NO3 )2 , primarily by eliciting cytokine production, in a cell type-dependent manner.

Multi walled carbon nanotubes (MWCNTs) are one of the most intensively explored nanomaterials because of their unique physical and chemical properties. Due to the widespread use of MWCNTs, it is important to investigate their effects on human health. The precise mechanism of MWCNT toxicity has not been fully elucidated. The present study was designed to examine the mechanisms of MWCNT toxicity toward human promyelocytic leukemia HL-60 cells. First, we found that MWCNTs decreased the viability of neutrophil-like differentiated HL-60 cells but not undifferentiated HL-60 cells. Because neutrophil-like differentiated HL-60 cells exhibit enhanced phagocytic activity, the cytotoxicity of MWCNTs is dependent on the intracellularly localized MWCNTs. Next, we revealed that the cytotoxicity of MWCNTs is correlated with the intracellular accumulation of iron that is released from the engulfed MWCNTs in an acidic lysosomal environment. The intracellular accumulation of iron was repressed by treatment with cytochalasin D, a phagocytosis inhibitor. In addition, our results indicated that iron overload enhanced the release of interleukin-8 (IL-8), a chemokine that activates neutrophils, and subsequently elevated intracellular calcium concentration ([Ca2+]i). Finally, we found that the sustained [Ca2+]i elevation resulted in the loss of mitochondrial membrane potential and the increase of caspase-3 activity, thereby inducing apoptotic cell death. These findings suggest that the iron overload caused by engulfed MWCNTs results in the increase of IL-8 production and the elevation of [Ca2+]i, thereby activating the mitochondria-mediated apoptotic pathway.

Oxidative stress plays a critical role in nanotoxicity. Various types of nanoparticles are known to induce oxidative stress by generating intracellular reactive oxygen species (ROS). Cellular uptake of nanoparticles and intracellular metal ion release are important factors for intracellular ROS generation. Besides, ROS generation can result from interactions of nanoparticles and cells that lead to mitochondrial dysfunction. In vivo, nanoparticles induce the secretion of cytokines, which, in turn, induce secondary oxidative stress via generation of ROS and free radicals. However, not all nanoparticles induce oxidative stress. Intracellular ROS generation by nanoparticles depends on their physical and chemical properties, such as the crystalline phase, adsorption ability, and solubility. Even if the particle size is nanoscale (1-100 nm), physically and chemically inactive particles do not induce oxidative stress. Thus, the particle size is not a direct influencing factor in nanoparticle-induced oxidative stress.

Nanoparticles (NPs) are promising products in industry and medicine due to their unique physicochemical properties. In particular, zinc oxide (ZnO) NPs are extensively incorporated into sunscreens to protect the skin from exposure to ultraviolet radiation. However, there are several health concerns about skin penetration and the resultant toxicity. As methodologies for evaluating NP toxicity are under development, it is difficult to fully assess the toxicity of ZnO NPs toward humans. In this study, we developed a platform to simultaneously detect skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs. First, we generated a stable reporter cell line expressing green fluorescent protein (GFP) under the control of interleukin-8 (IL-8) promoter activity. The expression levels of GFP induced by zinc reflected the endogenous IL-8 expression levels and the pro-inflammatory responses. Next, we found that fibrin hydrogel can reproduce permeability to zinc ion of a human skin equivalent model and is therefore a promising material to assess skin permeability to zinc ion. Then, we constructed a fibrin hydrogel-based in vitro bioassay system for the simultaneous detection of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs by using a stable reporter cell line and a fibrin hydrogel layer. This bioassay system is a promising in vitro permeation test due to its technical simplicity and good predictability. Overall, we believe that our bioassay system can be widely used in the cosmetics and pharmaceutical industries.

Properties of Lactobacillus plantarum group strains isolated from two kinds of Japanese post-fermented teas, Ishizuchi-kurocha and Awa-bancha, were compared. Although lactic acid bacteria isolated from the fermented teas were identified as L. plantarum via homology comparison of 16S ribosomal RNA gene sequences, classification of L. plantarum based on ribosomal proteins showed that the strains isolated from Ishizuchi-kurocha and Awa-bancha were different. According to classification by the ribosomal protein typing, Ishizuchi-kurocha-derived strains belong to the same group as L. plantarum subsp. plantarum JCM 1149T. Awa-bancha-derived strains were assigned to a different group. This pattern was also applicable to strains isolated more than 10 years ago. A further analysis based on recA and a dnaK gene showed that Awa-bancha-derived strains were closely related to L. pentosus. The interactions with cultured cells were different between strain JCM 1149T and the Ishizuchi-kurocha-derived strains. The Ishizuchi-kurocha-derived strains showed strong adhesion to Caco-2 cells. In contrast, strain JCM 1149T and the Awa-bancha-derived strains hardly adhered to Caco-2 cells. According to the ribosomal protein typing, sugar utilization, and interaction with Caco-2 cells, although these properties were dependent on the strain strictly speaking, the L. plantarum group strains in this study can be subdivided into two groups: (1) type strain JCM 1149T and Ishizuchi-kurocha-derived strains and (2) Awa-bancha-derived strains. A regionally unique microorganism may persist in each traditional fermented drink.

5-Hydroxy-4-phenyl-butenolide (5H4PB) is a bioactive compound with antifungal and anti-obesity properties. Although it has recently been shown that 5H4PB activates peroxisome proliferator-activated receptor-gamma (PPARγ), the effect of 5H4PB on intracellular signaling pathways has not been clarified. In this study, we found that 5H4PB activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway, which plays an important role in cellular defense against oxidative stress, and the subsequent upregulation of ARE-dependent cytoprotective genes, including the heme oxygenase-1, catalase, and superoxide dismutase genes, without exhibiting cytotoxicity. In addition, 5H4PB significantly attenuated intracellular ROS generation, glutathione oxidation, and DNA damage induced by hydrogen peroxide (H2O2) exposure in mouse fibroblast cells. Furthermore, we demonstrated that pretreatment with 5H4PB confers a significant cytoprotective effect against H2O2-induced cell death in mouse cultured fibroblasts and primary hepatocytes. Thus, our study demonstrated that 5H4PB enhanced cellular resistance to oxidative damage via activation of the Nrf2/ARE signaling pathway.

Among the crystal forms of calcium carbonate, aragonite has needle-like shape. Although materials with needle-shaped crystals are associated with pulmonary toxicity, the toxic activity of aragonite is unclear. Therefore, proinflammatory potential of aragonite, neutralized aragonite and potassium titanate whisker was evaluated. The cellular effects of aragonite were weaker than those of potassium titanate whisker. Aragonite treatment induced the expression of chemokines in A549 cells and macrophages. Although aragonite exhibited proinflammatory effects in vitro, pulmonary inflammation was not observed in vivo after intratracheal administration of aragonite in mice. We did not observe the induction of inflammatory cytokine secretion or tissue lesion in the lungs of mice after administration of aragonite. Potassium titanate whisker treatment induced chemokine secretion in vitro. An increase in the number of neutrophils was observed in the mice lung tissue after administration of potassium titanate whisker. Aragonite and neutralized aragonite both induced an increase in the levels of intracellular calcium, but the levels were significantly higher in cells treated with aragonite than in cells treated with neutralized aragonite. These results suggested that intracellular calcium release mediates the cellular effects of aragonite. The toxicity of aragonite based on its needle-like structure was also not observed.

Titanium dioxide (TiO2) nanoparticles are important industrial nano-objects with wide applications, including as photocatalysts and sunscreen components. Recently, the phototoxicity of TiO2 nanoparticles has been a concern. However, phototoxicity caused by photocatalytic activity may differ between anatase and rutile nanoparticles. In the present study, we compared the phototoxicity of anatase and rutile nanoparticles. Human keratinocyte HaCaT cells were treated with stable TiO2 nanoparticle suspensions. Without UVA irradiation, TiO2 nanoparticles did not affect mitochondrial activity or cell membranes. However, exposure to rutile nanoparticle suspensions inhibited cell growth and induced HO-1 gene expression without UVA irradiation. These effects may be explained by the hydrophobic surface of rutile nanoparticles. Next, TiO2-exposed cells were irradiated with UVA for 4 h and effects of TiO2 nanoparticles on cells were examined. The rutile nanoparticles did not show any cellular effects after UVA irradiation. However, the anatase nanoparticles caused strong phototoxicity. Decreased mitochondrial activity, cell membrane damage and the induction of oxidative stress were observed in the cells exposed to anatase nanoparticles with UVA irradiation. Cellular uptake of the nanoparticles was observed in both anatase- and rutile-exposed cells. These results suggest that internalized anatase nanoparticles are important for phototoxicity. Additionally, the exposure of a 3D skin model to TiO2 nanoparticles did not result in significant toxicity. In conclusion, rutile nanoparticles used in sunscreen did not exhibit phototoxic activity. Despite the strong phototoxic activity of anatase nanoparticles in cell cultures, they demonstrated no phototoxicity using a 3D skin model.

Effects of two kinds of multiwall carbon nanotubes (MWCNTs) on cells were examined. The effects of MWNT-7, which has been reported to be carcinogenic, and MWCNT-B, whose toxicity is unclear, were examined in both epithelial cells and macrophages. Human lung carcinoma A549 cells were used as representative epithelial cells and differentiated human monocyte THP-1 cells, as well as rat pulmonary macrophages NR8383, were employed to examine possible harmful effects of the MWCNTs. The MWCNTs induced the production of chemokines such as interleukin-8 (IL-8). MWCNTs were found to more strongly affect macrophages than epithelial cells. In addition, the toxicity was more pronounced in the MWNT-7 exposed cells than in those exposed to MWCNT-B. Cytochalasin D and amiloride treatment of differentiated THP-1 cells reduced cell-associated MWCNTs and IL-8 induction. To confirm these cellular influences in vivo, intratracheal administration of each type of MWCNT was performed by pharyngeal aspiration in the mouse lung. Analysis of bronchoalveolar lavage fluid (BALF) showed increase of inflammatory monocyte in MWNT-7 exposed animals at 1week after. In addition, neutrophils in the BALF were also significantly increased MWNT-7 exposed animals at 1 week and 1 month after. Aspiration of MWNT-7 caused formation of granulomas in the lung. Formation of the granulomas was not observed in the case of MWCNT-B. These results suggest that cellular uptake of the MWCNTs by phagocytosis and chemokine induction is important aspects of their toxicity.

Metal oxide nanoparticles have an industrial value, although their harmful effects are also known. Induction of respiratory inflammation through their inhalation is a serious indicator of their toxicity. Although the phenomenon of metal ion release is involved in the induction of inflammation, all metal ions are not necessarily toxic. However, currently, no particular index to evaluate cytotoxicity caused by nanoparticles exists. An index based on biological response is critical. In the present study, we examined the gene expression-based index for nanoparticle-derived cytotoxicity. The cellular effects of six kinds of metal oxide nanoparticles, ZnO, NiO, CuO, MgO, Bi2O3, and MoO3 on A549 cells were examined. It was seen that lactate dehydrogenase (LDH) assay, which is one of the most important assays for assessing cell membrane damage, is inhibited by metal ions released from the metal oxide nanoparticles. In some cases, enzyme activity-based assay was not suitable for the evaluation of cytotoxicity of nanoparticles. ZnO and CuO nanoparticles displayed severe cytotoxicity and enhanced gene expression of heme oxygenase-1 (HO-1) and interleukin-8 (IL-8). The IL-8 gene expression was also increased from Bi2O3 exposure. Additionally, the gene expression of metallothionein 2A (MT2A) was enhanced in the ZnO, CuO, and Bi2O3 exposed cells. These results suggest that these nanoparticles released metal ions in the cells. The enhancement of HO-1, IL-8, and MT2A gene expressions was related to the cytotoxic activity of metal oxide nanoparticles. Thus, the expression level of these genes is a good indicator of nanotoxicology of metal oxide nanoparticles.

Properties of Lactobacillus plantarum group strains isolated from two kinds of Japanese post-fermented teas, Ishizuchi-kurocha and Awa-bancha, were compared. Although lactic acid bacteria isolated from the fermented teas were identified as L. plantarum via homology comparison of 16S ribosomal RNA gene sequences, classification of L. plantarum based on ribosomal proteins showed that the strains isolated from Ishizuchi-kurocha and Awa-bancha were different. According to classification by the ribosomal protein typing, Ishizuchi-kurocha-derived strains belong to the same group as L. plantarum subsp. plantarum JCM 1149T. Awa-bancha-derived strains were assigned to a different group. This pattern was also applicable to strains isolated more than 10 years ago. A further analysis based on recA and a dnaK gene showed that Awa-bancha-derived strains were closely related to L. pentosus. The interactions with cultured cells were different between strain JCM 1149T and the Ishizuchi-kurocha-derived strains. The Ishizuchi-kurocha-derived strains showed strong adhesion to Caco-2 cells. In contrast, strain JCM 1149T and the Awa-bancha-derived strains hardly adhered to Caco-2 cells. According to the ribosomal protein typing, sugar utilization, and interaction with Caco-2 cells, although these properties were dependent on the strain strictly speaking, the L. plantarum group strains in this study can be subdivided into two groups: (1) type strain JCM 1149T and Ishizuchi-kurocha-derived strains and (2) Awa-bancha-derived strains. A regionally unique microorganism may persist in each traditional fermented drink.

Indium tin oxide (ITO) is widely used as a transparent conducting electrode in photoelectron devices. Because ITO production has soared, the potential health hazards caused by occupational exposure to this material have attracted much attention. However, little is known about the mechanisms of the toxic action of ITO nanoparticles (NPs). The present study was designed to examine the genotoxic mechanisms of ITO NPs using human lung epithelial A549 cells. We found that exposing A549 cells to ITO NPs triggered the intracellular accumulation of ITO NPs, the generation of reactive oxygen species (ROS), and the induction of DNA damage. Treatment of the cells with N-acetyl-l-cysteine (NAC), an ROS quenching agent, decreased intracellular ROS levels but not DNA damage, indicating that the genotoxic effect of ITO NPs is not mediated by intracellular ROS. Interestingly, treatment with ammonium chloride, a lysosomotropic agent, decreased intracellular solubility of ITO NPs and attenuated DNA damage. Nuclear accumulation of indium ions in ITO-NP-exposed cells was confirmed by inductively coupled plasma-mass spectrometry. Our results indicate that the ITO-NP-mediated genotoxicity is caused by indium ions that are solubilized in the acidic lysosomal condition and accumulated in the nucleus where they damage DNA, without the involvement of ROS.

Indium tin oxide (ITO) is widely used in liquid crystal displays (LCDs) or plasma and mobile phone displays. Elevated production and usage of ITO in such displays have led to increased concerns over the safety of industrial workers exposed to particulate aerosols produced during cutting, grinding and polishing of these materials. However, the cellular effects of ITO nanoparticles (NPs) are still unclear, although it has been reported that micro-scale ITO particles induce cytotoxicity. The aim of this study was to examine the potential of ITO NPs to induce cytotoxicity, oxidative stress, and DNA damage using human lung adenocarcinoma A549 cells. Here, stable dispersions of a medium containing ITO NPs were obtained using pre-adsorption and centrifugal fractionation methods, and the A549 cells were incubated in this medium. The ITO NPs showed low cytotoxic effects as shown by the WST-1 and LDH assays. Transmission electron microscopy observations showed the cellular uptake of ITO NPs. The ITO NPs increased the intracellular level of reactive oxygen species and the expression of the heme oxygenase 1 gene. Further, the results of alkaline comet assays showed that ITO NPs induced DNA damage. Thus, these results suggest that ITO NPs possess a genotoxic potential on human lung adenocarcinoma A549 cells.

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO3)-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO3 particles.

Nonalcoholic steatohepatitis (NASH) is a common hepatic manifestation of metabolic syndrome and can lead to hepatic cirrhosis and cancer. It is considered that NASH is caused by multiple parallel events, including abnormal lipid metabolism, gut-derived-endotoxin-induced inflammation and adipocytokines derived from adipose tissue, suggesting that other tissues are involved in NASH development. Previous studies demonstrated that spleen enlargement is observed during the course of NASH pathogenesis. However, the involvement of splenic status in the progression of NASH remains unclear. In this study, we examined hepatic and splenic histopathological findings in the early stage of NASH using the Tsumura Suzuki Obese Diabetes (TSOD) mouse model established for assessing NASH. We found that 12-week-old TSOD mice clearly exhibited the histopathological features of NASH in the early stage. At this age, the spleen of TSOD mice showed markedly higher iron level than that of control Tsumura Suzuki Non Obesity (TSNO) mice. The level of accumulated iron was significantly decreased by feeding a diet with glucosyl hesperidin, a bioactive flavonoid, accompanied with alleviation of hepatic lesions. Furthermore, we found that splenic iron level was positively correlated with the severity of NASH manifestations, suggesting that abnormalities in the spleen are involved in the development of NASH.

It has been proposed that selenium, an element chemically similar to sulfur, can participate in some of the same biological pathways as sulfur, although only a few studies have been confirmed this. In this study, we investigated the relationship between selenium and sulfur-dependent luminescence in Vibrio fischeri. The luminescence of V. fischeri was induced by the addition of sulfur-containing compounds such as Na₂SO₄ and L-cystine, and their luminescence was suppressed, in a dose-dependent manner, by the addition of the selenium-containing compounds Na₂SeO₄ and L-selenocystine. Since the viability of V. fischeri was not affected by the addition of low concentration of selenium-containing compounds, the decrease in luminescence intensity cannot be explained by cell death. Kinetic analysis performed using Lineweaver-Burk plots demonstrate that Na₂SeO₄ and L-selenocystine act as competitive suppressors in inorganic sulfur (Na₂SeO₄)-dependent luminescence. In contrast, these selenium-containing compounds act as uncompetitive suppressors in organic sulfur (L-cystine)-dependent luminescence.

Bioluminescence is emitted by various living organisms, including bacteria. While the induction mechanism in marine luminescent bacteria, such as Vibrio fischeri and V. harveyi, has been well characterized, this mechanism has not been studied in detail in the non-marine luminescent bacterium Photorhabdus luminescens. Therefore, we investigated the effect of cations and anions on the induction of luminescence by P. luminescens. Cultivation of cells in an inorganic salts solution (ISS) containing KCl, CaCl2, MgCl2, NaHCO3, and MgSO4 resulted in a rapid increase in luminescence intensity. Moreover, the induction of luminescence in the ISS medium was not dependent on cell density, since cell densities remained unchanged during 48 h. Furthermore, we found that compounds containing K+, Mg2+, and HCO3– were necessary to induce cell density-independent luminescence. The intensity of luminescence per cell cultured in medium containing KCl, MgCl2, and NaHCO3 was approximately 100-fold higher than that cultured in NB. In contrast, when cells actively grew in normal growth condition, the intensity of luminescence per cell was not increased even in the presence of K+, Mg2+, and HCO3–. Thus, these results suggest that the luminescence of P. luminescens is regulated by 2 independent cell density-dependent and -independent mechanisms.

In spite of its central importance in research efforts, the relationship between seawater compounds and bacterial luminescence has not previously been investigated in detail. Thus, in this study, we investigated the effect of cations (Na(+) , K(+) , NH(4) (+) , Mg(2+) , and Ca(2+) ) and anions (Cl(-) , HCO(3) (-) , CO(3) (2-) , and NO(3) (-) ) on the induction of both inorganic (sulfate, sulfite, and thiosulfate) and organic (L-cysteine and L-cystine) sulfur-dependent luminescence in Vibrio fischeri. We found that HCO(3) (-) (bicarbonate) and CO(3) (2-) (carbonate), in the form of various compounds, had a stimulatory effect on sulfur-dependent luminescence. The luminescence induced by bicarbonate was further promoted by the addition of magnesium. Potassium also increased sulfur-dependent luminescence when sulfate or thiosulfate was supplied as the sole sulfur source, but not when sulfite, L-cysteine, or L-cystine was supplied. The positive effect of potassium was accelerated by the addition of magnesium and/or calcium. Furthermore, the additional supply of magnesium improved the induction of sulfite- or L-cysteine-dependent luminescence, but not the l-cystine-dependent type. These results suggest that sulfur-dependent luminescence of V. fischeri under nutrient-starved conditions is mainly controlled by bicarbonate, carbonate, and potassium. In addition, our results indicate that an additional supply of magnesium is effective for increasing V. fischeri luminescence.

Synechocystis sp. PCC 6803 exhibits light-activated heterotrophic growth (LAHG) under dark conditions with glucose as a carbon source. The light activation is remarkable at a late period of photoautotrophic preculture, such as the late-linear and stationary growth phases. To understand the physiological effects of light irradiation and glucose under LAHG conditions, their effects on the expression of soluble proteins were analyzed by means of 2D-PAGE. Various soluble proteins, which were minimal under photoautotrophic preculture conditions, were observed clearly under LAHG conditions, suggesting that proteins were synthesized actively under these conditions. Fructose 1,6-bisphosphate aldolase, one of the glycolytic enzymes, was found to be induced under LAHG conditions on 2D-PAGE. The activity of fructose 1,6-bisphosphate aldolase, which had decreased during photoautotrophic preculture, also increased under LAHG conditions, similar to the mRNA level of the encoding gene, fbaA. In addition, we found that a deletion mutant of sll1330, a putative gene containing a helix-turn-helix DNA-binding motif, could not grow under LAHG conditions, whereas it could grow photoautotrophically. The increases in the protein level of FbaA and fbaA gene expression observed in wild-type cells under LAHG conditions were greatly inhibited in the deletion mutant. These results suggest that the regulation of fbaA gene expression by way of sll1330 is one of the important processes in Synechocystis sp. PCC 6803 under light pulse LAHG conditions.

In the complete annotated genome sequences of cyanobacterium Synechocystis sp. PCC 6803, one can find many putative genes for two-component response regulators that include a helix-turn-helix DNA-binding domain. The mRNA level of one of the putative genes, sll1330, was increased by glucose, especially in the presence of light. We successfully disrupted the sll1330 gene by targeted mutagenesis with a spectinomycin resistance cassette. Deltasll1330 could not grow well under light-activated heterotrophic growth conditions. Analyses of the expression of glycolytic genes revealed that the mRNA levels of five glycolytic genes, that is, glk (sll0593), pfkA (sll1196), fbaA (sll0018), gpmB (slr1124), and pk (sll0587), were decreased, and were regulated by Sll1330 under light and glucose-supplemented conditions. The Synechocystis sp. PCC 6803 genome each encodes two isozymes for these five glycolytic genes, suggesting that each of the two isozymes is regulated by Sll1330 at the mRNA level.

Due to the widespread use of indium tin oxide (ITO), it is important to investigate its effect on human health. In this study, we evaluated the cellular effects of ITO nanoparticles (NPs), indium chloride (InCl3) and tin chloride (SnCl3) using human lung epithelial A549 cells. Transmission electron microscopy and inductively coupled plasma mass spectrometry were employed to study cellular ITO NP uptake. Interestingly, greater uptake of ITO NPs was observed, as compared with soluble salts. ITO NP species released could be divided into two types: 'indium release ITO' or 'tin release ITO'. We incubated A549 cells with indium release ITO, tin release ITO, InCl3 or SnCl2 and investigated oxidative stress, proinflammatory response, cytotoxicity and DNA damage. We found that intracellular reactive oxygen species were increased in cells incubated with indium release ITO, but not tin release ITO, InCl3 or SnCl2. Messenger RNA and protein levels of the inflammatory marker, interleukin-8, also increased following exposure to indium release ITO. Furthermore, the alkaline comet assay revealed that intracellular accumulation of indium ions induced DNA damage. Our results demonstrate that the accumulation of ionic indium, but not ionic tin, from ITO NPs in the intracellular matrix has extensive cellular effects.

It has been proposed that selenium, an element chemically similar to sulfur, can participate in some of the same biological pathways as sulfur, although only a few studies have been confirmed this. In this study, we investigated the relationship between selenium and sulfur-dependent luminescence in Vibrio fischeri. The luminescence of V. fischeri was induced by the addition of sulfur-containing compounds such as Na₂SO₄ and L-cystine, and their luminescence was suppressed, in a dose-dependent manner, by the addition of the selenium-containing compounds Na₂SeO₄ and L-selenocystine. Since the viability of V. fischeri was not affected by the addition of low concentration of selenium-containing compounds, the decrease in luminescence intensity cannot be explained by cell death. Kinetic analysis performed using Lineweaver-Burk plots demonstrate that Na₂SeO₄ and L-selenocystine act as competitive suppressors in inorganic sulfur (Na₂SeO₄)-dependent luminescence. In contrast, these selenium-containing compounds act as uncompetitive suppressors in organic sulfur (L-cystine)-dependent luminescence.

Bioluminescence is emitted by various living organisms, including bacteria. While the induction mechanism in marine luminescent bacteria, such as Vibrio fischeri and V. harveyi, has been well characterized, this mechanism has not been studied in detail in the non-marine luminescent bacterium Photorhabdus luminescens. Therefore, we investigated the effect of cations and anions on the induction of luminescence by P. luminescens. Cultivation of cells in an inorganic salts solution (ISS) containing KCl, CaCl2 , MgCl2 , NaHCO3 , and MgSO4 resulted in a rapid increase in luminescence intensity. Moreover, the induction of luminescence in the ISS medium was not dependent on cell density, since cell densities remained unchanged during 48 h. Furthermore, we found that compounds containing K(+) , Mg(2+) , and HCO3(-) were necessary to induce cell density-independent luminescence. The intensity of luminescence per cell cultured in medium containing KCl, MgCl2 , and NaHCO3 was approximately 100-fold higher than that cultured in NB. In contrast, when cells actively grew in normal growth condition, the intensity of luminescence per cell was not increased even in the presence of K(+) , Mg(2+) , and HCO3(-) . Thus, these results suggest that the luminescence of P. luminescens is regulated by 2 independent cell density-dependent and -independent mechanisms.

In this study, we investigated the relationship between MgSO(4) and luminescence in Vibrio fischeri under nutrient-starved conditions. When V. fischeri was cultured in an artificial seawater medium, the luminescence intensity was low relative to that observed under normal growth conditions. It decreased during the initial 14 h, and then increased slightly at 24 h. This regulation of luminescence was not dependent on the quorum-sensing mechanism, because the cell densities had not reached a critical threshold concentration. Under MgSO(4)-starved conditions, luminescence was not fully induced at 14 h, and decreased at 24 h. In contrast, induction of luminescence occurred under MgSO(4)-supplemented conditions, but MgSO(4) alone was insufficient to induce luminescence, and required NaHCO(3) or KCl. These results suggest that the luminescence of V. fischeri is controlled by an exogenous sulfur source under nutrient-starved conditions. In addition, they indicate that the induction of sulfur-dependent luminescence is regulated by the NaHCO(3) or KCl concentration.

Despite the universal requirement for sulfur in living organisms, it is not known whether the luminescence of Vibrio fischeri is sulfur-dependent and how sulfur affects the intensity of its luminescence. In this study, we investigated the requirement for sulfur in V. fischeri luminescence under nutrient-starved conditions. Full induction of V. fischeri luminescence required MgSO4; in artificial seawater cultures that lacked sufficient MgSO4, its luminescence was not fully induced. This induction of luminescence was not dependent on autoinduction because the cell density of V. fischeri did not reach the critical threshold concentration. In addition to MgSO4, this cell density-independent luminescence was induced or maintained by nontoxic concentrations of l-cysteine, sulfate, sulfite, and thiosulfate. Moreover, the addition of N -3-oxo-hexanoyl homoserine lactone and N -octanoyl homoserine lactone, which are known autoinducers in V. fischeri, did not induce luminescence under these conditions. This result suggested that the underlying mechanism of luminescence may be different from the known autoinduction mechanism. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Molecular analyses of 16S ribosomal RNA genes (rDNA) revealed that microbial communities in the rhizosphere are highly complex. To systematically characterize the cell size of the bacteria in the rhizosphere, we selected bacteria that potentially could be passed through 0.22- or 0.45-microm-pore-size filters and then PCR amplified the 16S rDNA genes using the universal primer pairs 27F/1492R and 63F/1387R. The PCR-amplified rDNAs extracted from bacteria that had been passed through 0.45-microm-pore-size filters could be detected in agarose gels after electrophoresis; whereas after filtration of the bacteria through 0.22-microm-pore-size filters no PCR-amplified rDNAs were found. Comparison of random cloning and sequencing of the libraries of the PCR-amplified rDNAs with or without cell size selection showed that bacteria belonging to the candidate phylogenic divisions of OD1 (OP11-derived 1), OP11, TM7, and OP5 can be concentrated by cell size selection using 0.45-microm-pore-size filters, but not by using 0.22-microm-pore-size filters. OD1, OP11, TM7 and OP5 bacteria have yet to be cultivated; therefore, our concentration method may be used as an initial step in studies to analyze the structural properties of OD1, OP11, and TM7 bacteria.