Itaru Yanagihara

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.

Roberts syndrome is caused by mutations in ESCO2, a human homolog of yeast ECO1 that is essential for the establishment of sister chromatid cohesion

Roberts syndrome is an autosomal recessive disorder characterized by craniofacial anomalies, tetraphocomelia and loss of cohesion at heterochromatic regions of centromeres and the Y chromosome. We identified mutations in a new human gene, ESCO2, associated with Roberts syndrome in 15 kindreds. The ESCO2 protein product is a member of a conserved protein family that is required for the establishment of sister chromatid cohesion during S phase and has putative acetyltransferase activity.

Placental Features of Chorioamnionitis Colonized With Ureaplasma Species in Preterm Delivery

Ureaplasma spp. is detected in the urogenital tract, including the vagina, cervix, chorioamnion, and placenta. Their colonization is associated with histologic chorioamnionitis (CAM), often observed in placentas from preterm delivery. We isolated Ureaplasma spp. from 63 preterm placentas among 151 specimens, which were delivered at <32 wk of gestation. Of the 63 placentas, 52 (83%) revealed CAM in cultures positive for Ureaplasma spp., however, CAM was observed only in 30% (26/88) of cultures negative for Ureaplasma spp. (p < 0.01). Colonization by Ureaplasma spp. was an independent risk factor for CAM (OR, 11.27; 95% CI, 5.09–24.98). Characteristic neutrophil infiltration was observed in the amnion and subchorion (bistratified pattern) in cultures positive for Ureaplasma spp. FISH analysis of CAM placenta with male infant pregnancy indicated that bistratified infiltrated neutrophils showed the XX karyotype and umbilical vein infiltrated neutrophils showed XY karyotype. The distribution of sulfoglycolipid, the receptor of Ureaplasma spp., was mainly detected in the amnion. Ureaplasmal urease D protein and ureB gene were both detected in the amnion, indicating direct colonization by Ureaplasma spp.

Amino Acid Esters Prevent Thermal Inactivation and Aggregation of Lysozyme

Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat‐induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 °C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na‐phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 °C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat‐induced aggregation.

Structure and Functional Characterization of Vibrio parahaemolyticus Thermostable Direct Hemolysin

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramer forms a central pore with dimensions of 23 Å in diameter and ∼50 Å in depth. π-Cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

Analysis of the stable levels of messenger RNA derived from different polymorphic alleles in the vitamin D receptor gene

Abstract: The association between polymorphisms in the vitamin D receptor (VDR) gene and bone mineral density (BMD) has been studied by many investigators. However, the question of how polymorphisms in the gene modulate the function of the VDR remains to be answered. To address this issue, we examined the mRNA levels of the VDR in relation to polymorphisms. First, we compared the levels of mRNA between the allele with the polymorphic TaqI-digestive site (t) and nondigestive site (T) located at exon 9 of the VDR gene determined by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from peripheral mononuclear cells in volunteers whose genotype is Tt. After the amplification of cDNA by PCR, the amplified fragments were digested by TaqI. The digested (t) and undigested (T) fragments were visualized by ethidium bromide and semiquantified by an image analyzer. In 24 subjects, the mRNA levels of allele t were significantly higher than those of allele T (1.35 fold, P < 0.001). Second, the VDR mRNA levels were estimated by competitive RT-PCR in 60 healthy subjects (35 TT, 24 Tt, 1 tt). The competitive template was 47 bases shorter than the product of the wild-type gene. After RT-PCR, the mRNA level was determined by a comparison with the competitive fragments. No significant difference in the mRNA level was observed between two groups (1.75 ± 0.84 and 1.65 ± 0.99 10−13 mol/g total RNA in TT and Tt, respectively). In addition, no significant relationship was observed between the VDR mRNA levels and BMD in the 23 subjects whose BMD data were available. In conclusion, higher mRNA levels of allele t than T were detected, but the difference did not result in higher levels of VDR mRNA in subjects with the Tt genotype compared to those with the TT genotype.

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.

Effects of Ureaplasma parvum lipoprotein multiple-banded antigen on pregnancy outcome in mice

Ureaplasma spp. are members of the family Mycoplasmataceae and have been considered to be associated with chorioamnionitis and preterm delivery. However, it is unclear whether Ureaplasma spp. have virulence factors related to these manifestations. The purpose of the present study was to determine whether the immunogenic protein multiple-banded antigen (MBA) from Ureaplasma parvum is a virulence factor for preterm delivery. We partially purified MBA from a type strain and clinical isolates of U. parvum, and also synthesized a diacylated lipopeptide derived from U. parvum, UPM-1. Using luciferase assays, both MBA-rich fraction MRF and UPM-1 activated the NF-κB pathway via TLR2. UPM-1 upregulated IL-1β, IL-6, IL-12p35, TNF-α, MIP2, LIX, and iNOS in mouse peritoneal macrophage. MRF or UPM-1 was injected into uteri on day 15 of gestation on pregnant C3H/HeN mice. The intrauterine MRF injection group had a significantly higher incidence of intrauterine fetal death (IUFD; 38.5%) than the control group (14.0%). Interestingly, intrauterine injection of UPM-1 caused preterm deliveries at high concentration (80.0%). In contrast, a low concentration of UPM-1 induced a significantly higher rate of fetal deaths (55.2%) than the control group (14.0%). The placentas of the UPM-1 injection group showed neutrophil infiltration and increased iNOS protein expression. Our data indicate that MBA from the clinical isolate of U. parvum is a potential virulence factor for IUFD and preterm delivery in mice and that the N-terminal diacylated lipopeptide is essential for the initiation of inflammation.

E2A and CBP/p300 Act in Synergy To Promote Chromatin Accessibility of the Immunoglobulin κ Locus

V(D)J recombination of Ig and TCR genes is strictly regulated in a lineage- and stage-specific manner by the accessibility of target gene chromatin to the recombinases RAG1 and RAG2. It has been shown that enforced expression of the basic helix–loop–helix protein, E2A, together with RAG1/2 in a nonlymphoid cell line BOSC23 can induce V(D)J recombination in endogenous Igκ and TCR loci by increasing chromatin accessibility of target gene segments. In this study, we demonstrate that ectopically expressed E2A proteins in BOSC23 cells have the ability to bind directly to the promoter and recombination signal sequence of Vκ genes and to recruit histone acetyltransferase CBP/p300. Overexpression of CBP/p300 in conjunction with E2A results in enhancement of E2A-induced histone acetylation, germline transcription, and Igκ rearrangement. Conversely, knockdown of endogenous CBP/p300 expression by small interfering RNA leads to a decrease in histone acetylation, germline transcription and Igκ rearrangement. Furthermore, analyses using a mouse pre-B cell line revealed that endogenous E2A proteins also bind to a distinct set of Vκ genes and regulatory regions in the mouse Igκ locus and act to increase histone acetylation by recruiting p300, confirming the similar findings observed with BOSC23 cells. These observations indicate that E2A plays critical roles in inducing Igκ rearrangement by directly binding to and increasing chromatin accessibility at target gene segments.

EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein

The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far‐UV and near‐UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes α‐helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side‐chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8‐anilinonaphthalene‐1‐sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea‐unfolding transitions, monitored by far‐UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB 15N‐1H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea‐unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.