Yuichi Kawai

Silica and titanium dioxide nanoparticles cause pregnancy complications in mice

The increasing use of nanomaterials has raised concerns about their potential risks to human health. Recent studies have shown that nanoparticles can cross the placenta barrier in pregnant mice and cause neurotoxicity in their offspring, but a more detailed understanding of the effects of nanoparticles on pregnant animals remains elusive. Here, we show that silica and titanium dioxide nanoparticles with diameters of 70 nm and 35 nm, respectively, can cause pregnancy complications when injected intravenously into pregnant mice. The silica and titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain. Mice treated with these nanoparticles had smaller uteri and smaller fetuses than untreated controls. Fullerene molecules and larger (300 and 1,000 nm) silica particles did not induce these complications. These detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side, and are abolished when the surfaces of the silica nanoparticles are modified with carboxyl and amine groups.

Carbon Nanotubes Elicit DNA Damage and Inflammatory Response Relative to Their Size and Shape

Carbon nanotubes (CNTs) have been one of the most extensively researched and developed nanomaterials. However, little concern has been placed on their safety. The biological effects of CNTs are believed to differ relative to size and shape. Thus, the relationship between the characteristics of CNTs and their safety needs to be evaluated. In this study, we examined the biological effects of different-sized multi-walled CNTs (MWCNTs) and single-walled CNTs (SWCNTs). Long and thick MWCNTs induced the strongest DNA damage while similar SWCNTs caused little effect. Comparison of inflammatory responses of various types of CNTs found that peritoneal CNT administration of long and thick MWCNTs increased the total cell number in abdominal lavage fluid in mice. These results indicate that long and thick MWCNT, but not short and thin MWCNT, cause DNA damage and severe inflammatory effects. These findings might provide useful information for constructing novel CNTs with safety.

Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid

The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast‐like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6‐overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild‐type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule‐associated protein 2 (MAP2)‐expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash‐1, the neuronal development‐related gene Wnt‐1, the neuron‐specific cell adhesion molecule N‐cadherin, and the neuron‐specific protein MAP2, resulting in neurogenesis. Moreover, E‐cadherin, a major cell adhesion molecule of wild‐type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.

Suppression of Sox6 in P19 cells leads to failure of neuronal differentiation by retinoic acid and induces retinoic acid-dependent apoptosis.

The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia, and fibroblast‐like cells. Sox6 mRNA increases rapidly in P19 cells during RA induction and then decreases during differentiation into neuronal cells. To investigate whether Sox6 expression is essential for neuronal differentiation, we established Sox6‐suppressed P19 (P19[anti‐Sox6]) cells by transfection of antisense‐Sox6 cDNA. Most of the P19[anti‐Sox6] cells showed no neurites and were not stained by the anti‐MAP 2 antibody, while the suppression of Sox6 expression nearly totally blocked neuronal differentiation in P19 cells. Further, Sox6 suppression caused RA‐dependent apoptosis by P19[anti‐Sox6] cells: RA‐treated P19[anti‐Sox6] cells showed chromatin condensation, DNA fragmentation, and an increase in caspase‐3‐like activity. Thus, Sox6 is considered essential for neuronal differentiation and may play an important role in the early stages of neuronal differentiation or apoptosis.

The use of a mutant TNF-α as a vaccine adjuvant for the induction of mucosal immune responses

Safe and potent adjuvants are required in order to establish effective mucosal vaccines. Cytokines are promising adjuvants because they are human-derived safe biomaterial and display immune-modulating functions. We have created a mutant tumor necrosis factor-alpha (TNF-alpha), mTNF-K90R, that exhibits high bioactivity and resistance to proteases. Here, we examined the potential of mTNF-K90R as a mucosal adjuvant. Initially, we showed that intranasal co-administration of mTNF-K90R with ovalbumin (OVA) potently produced OVA-specific Immunoglobulin (Ig) G antibodies (Abs) in serum and IgA Abs both at local and distal mucosal sites compared to co-administration with wild-type TNF-alpha. The OVA-specific immune response was characterized by high levels of serum IgG1 and increased production of interleukin-4 (IL-4), IL-5 and IL-10 from splenocytes of immunized mice, suggesting a Th2 response. Furthermore, intranasal immunization with an antigen from influenza virus plus mTNF-K90R exhibited mucosal adjuvant activity for induction of both systemic and mucosal immune responses. Importantly, histopathological examination of the nasal tissue of mTNF-K90R treated mice detected no signs of toxicity. These findings suggest that mTNF-K90R is safe and effective mucosal adjuvant and this system may have potential application as a universal mucosal adjuvant system for mucosal vaccines improving the immune response to a variety of viral antigens.

Short-term changes in intracellular ROS localisation after the silver nanoparticles exposure depending on particle size

Highlights • Exposing cells to nanosilver particles (AgNPs) immediately induces ROS.• Smaller AgNPs induce mitochondrial ROS production.• AgNP-induced mitochondrial ROS are independent of particle internalisation.

TNF superfamily member, TL1A, is a potential mucosal vaccine adjuvant

The identification of cytokine adjuvants capable of inducing an efficient mucosal immune response against viral pathogens has been long anticipated. Here, we attempted to identify the potential of tumor necrosis factor superfamily (TNFS) cytokines to function as mucosal vaccine adjuvants. Sixteen different TNFS cytokines were used to screen mucosal vaccine adjuvants, after which their immune responses were compared. Among the TNFS cytokines, intranasal immunization with OVA plus APRIL, TL1A, and TNF-alpha exhibited stronger immune response than those immunized with OVA alone. TL1A induced the strongest immune response and augmented OVA-specific IgG and IgA responses in serum and mucosal compartments, respectively. The OVA-specific immune response of TL1A was characterized by high levels of serum IgG1 and increased production of IL-4 and IL-5 from splenocytes of immunized mice, suggesting that TL1A might induce Th2-type responses. These findings indicate that TL1A has the most potential as a mucosal adjuvant among the TNFS cytokines.

Inhibitory effect of quinolone antimicrobial and nonsteroidal anti-inflammatory drugs on a medium chain acyl-CoA synthetase

The inhibitory effects of quinolone antimicrobial agents and nonsteroidal anti-inflammatory drugs on purified mouse liver mitochondrial medium chain acyl-CoA synthetase catalyzing the first reaction of glycine conjugation were examined, using hexanoic acid as a substrate. Enoxacin, ofloxacin, nalidixic acid, diflunisal, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid, which do not act as substrates, were potent inhibitors. Diflunisal, nalidixic acid, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid inhibited competitively this medium chain acyl-CoA synthetase with K(i) values of 0.6, 12.4, 19.6, 13.4, and 15.0 microM, respectively. Enoxacin and ofloxacin inhibited this medium chain acyl-CoA synthetase in a mixed-type manner with K(i) values of 23.7 and 38.2 microM, respectively. Felbinac, which is a substrate, inhibited the activity of this medium chain acyl-CoA synthetase for hexanoic acid (IC50 = 25 microM). The concomitant presence of enoxacin and felbinac strongly inhibited this medium chain acyl-CoA synthetase. These findings indicate that medium chain acyl-CoA synthetases may be influenced by quinolone antimicrobial and nonsteroidal anti-inflammatory drugs.

Presence of β-citryl-L-glutamic acid in the lens : its possible role in the differentiation of lens epithelial cells into fiber cells

The beta-CG concentration in the chicken brain was high during embryonic development and decreased rapidly to a lower level close to hatching, while the concentration in the eyeball which was also high during the embryonic life retained a fairly high level after hatching. The distribution of beta-CG in the bovine eye was determined. About 95% of total beta-CG content in the whole eye was localized in the lens. However, the distribution of beta-CG in the eye varied depending on species. beta-CG was exclusively localized in the lens in the eyes of fish and mammals, but distributed in both lens and retina in frogs. The molecule was localized in the retina rather than the lens in the chicken eye, although the concentrations was extremely low compared to those in the mammalian, amphibian and fish eyes. It was found that beta-CG is present ubiquitously in the lens or retina in various species. The distribution of beta-CG in the bovine lens was determined in the three cortex regions and nucleus. beta-CG was present at the highest concentration in the equatorial cortex, at a moderate concentration in the posterior and anterior cortex, and at the lowest concentration in the nucleus. Similar distribution patterns were also found in the rabbit and rat lens. When embryonic chick lens epithelial cells were cultured in the presence of fetal calf serum, the cells elongated, differentiated into fiber cells and formed lentoid bodies. The cells of lentoid bodies were stained strongly by the anti-beta-CG antibody, while cells around the structures were not. In addition, the beta-CG content in the lenses from the galactose cataractous rat decreased to about 20-30% of that in the normal lens. These findings suggest that beta-CG may play a role in the differentiation of epithelial cells into fiber cells.

Purification and properties of ß-citryl-l-glutamate-hydrolysing enzyme from rat testis particulate

beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiffs-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.