Effects of gestational exposure to 1.95‐GHz W‐CDMA signals for IMT‐2000 cellular phones: Lack of embryotoxicity and teratogenicity in rats

The present study was designed to evaluate whether gestational exposure to an EMF targeting the head region, similar to that from cellular phones, might affect embryogenesis in rats. A 1.95‐GHz wide‐band code division multiple access (W‐CDMA) signal, which is one applied for the International Mobile Telecommunication 2000 (IMT‐2000) system and used for the freedom of mobile multimedia access (FOMA), was employed for exposure to the heads of four groups of pregnant CD(SD) IGS rats (20 per group) for gestational days 7–17. The exposure was performed for 90 min/day in the morning. The spatial average specific absorption rate (SAR) for individual brains was designed to be 0.67 and 2.0 W/kg with peak brain SARs of 3.1 and 7.0 W/kg for low (group 3) and high (group 4) exposures, respectively, and a whole‐body average SAR less than 0.4 W/kg so as not to cause thermal effects due to temperature elevation. Control and sham exposure groups were also included. At gestational day 20, all dams were killed and fetuses were taken out by cesarean section. There were no differences in maternal body weight gain. No adverse effects of EMF exposure were observed on any reproductive and embryotoxic parameters such as number of live (243–271 fetuses), dead or resorbed embryos, placental weights, sex ratios, weights or external, visceral or skeletal abnormalities of live fetuses. Bioelectromagnetics 30:205–212, 2009. © 2008 Wiley‐Liss, Inc.     

Molecular Basis for E-cadherin Recognition by Killer Cell Lectin-like Receptor G1 (KLRG1)

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca²⁺-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K_d ∼7–12 μm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1⁺NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.Natural killer (NK)³ cells play a critical role in the innate immune system because of their ability to kill other cells. For example, NK cells can kill virus-infected cells and tumor cells without presensitization to a specific antigen, and they produce various cytokines, including interferon-γ and tumor necrosis factor-α (1). NK cells are controlled by both inhibitory and activating receptors that are expressed on their surfaces (2). The killer cell Ig-like receptor, Ly49, CD94/NKG2, and paired Ig-like type 2 receptor families include both inhibitory and activating members and thus are designated as paired receptor families. On the other hand, some inhibitory receptors, including KLRG1 (killer cell lectin-like receptor G1), and activating receptors, such as NKG2D, also exist. The integration of the signals from these receptors determines the final functional outcome of NK cells.These inhibitory and activating receptors can also be divided into two structurally different groups, the Ig-like receptors and the C-type lectin-like receptors, based on the structural aspects of their extracellular regions. The Ig-like receptors include killer cell Ig-like receptors and the leukocyte Ig-like receptors, and the C-type lectin-like receptors include CD94/NKG2(KLRD/KLRC), Ly49(KLRA), NKG2D(KLRK), NKR-P1(KLRB), and KLRG1. Many of these immune receptors recognize major histocompatibility complex class I molecules or their relatives (2–4), but there are still many orphan receptors expressed on NK cells. KLRG1 was one such orphan receptor; however, E-cadherin was recently found to be a ligand of KLRG1 (5, 6). Although major histocompatibility complex-receptor interactions have been extensively examined, the molecular basis of non-major histocompatibility complex ligand-receptor recognition is poorly understood.KLRG1 is a type II membrane protein, with one C-type lectin domain in the extracellular region, one transmembrane region, and one immunoreceptor tyrosine-based inhibitory motif. KLRG1 is expressed on a subset of mature NK cells in spleen, lungs, and peripheral blood during normal development. KLRG1 expression is induced on the surface of NK cells during viral responses (7, 8). NK cells expressing KLRG1 produce low levels of interferon-γ and cytokines and have a slow in vivo turnover rate and low proliferative responsiveness to interleukin-15 (9). Furthermore, KLRG1 is recognized as a marker of some T cell subsets, as follows. KLRG1 defines a subset of T cells, short lived effector CD8 T cells (SLECs), which are mature effector cells that express high levels of KLRG1 and cannot be differentiated into long lived memory CD8 T cells. In addition, memory precursor effector cells express low levels of KLRG1 and harbor the potential to become long lived memory CD8 T cells (10). Since SLECs exhibit stronger effector function than memory precursor effector cells, it is potentially beneficial, in terms of preventing harmful excess cytotoxicity, that SLECs express KLRG1 at a higher level to inhibit the immune response. Taken together, the expression of KLRG1 during the viral response and normal development might confer the inhibition of effector function and the regulation of NK and T cell proliferation (9).E-cadherin plays a pivotal role in Ca²⁺-dependent cell-cell adhesion and also contributes to tissue organization and development (11–14). E-cadherin is primarily expressed on epithelial cells, and its extracellular region consists of several domains that include cadherin motifs (15, 16). These domains mediate Ca²⁺-dependent homophilic interactions to facilitate cell adhesion. When E-cadherins form cis- or trans-homodimers, they utilize their N-terminal regions as an interface, which can dock with domain 1 of another E-cadherin to form strand exchange (17). Therefore, the N-terminal region plays important roles in homophilic binding and cell adhesion.KLRG1 recognizes E-cadherins (and other class I cadherins), which are widely expressed in tissues and form tight adhesive cell-cell junctions, and Ito et al. (5) demonstrated that E-cadherin binding by KLRG1 inhibits NK cytotoxicity. Further, Gründermann et al. (6) showed that the E-cadherin-KLRG1 interaction inhibits the antigen-induced proliferation and induction of the cytolytic activity of CD8 T cells. Therefore, it is plausible that E-cadherin recognition by KLRG1, expressed on the surfaces of NK cells and T cells, may raise their activation thresholds by transducing inhibitory signals. Such an inhibition would prevent the excess injury of normal cells, which might result in inflammatory autoimmune diseases. KLRG1 may also have an important role in monitoring and removing cancer cells that lose E-cadherin expression. A recent report demonstrated that N-terminal domains 1 and 2 of E-cadherin are critical for KLRG1 recognition (18); however, despite accumulating evidence supporting the functional importance of the E-cadherin-KLRG1 interaction, the molecular basis of this interaction is poorly understood. Here, we report that the N-terminal region of E-cadherin, comprising the dimer interface, is the binding site for KLRG1. This suggests that KLRG1 does not recognize the dimeric form of E-cadherin but rather recognizes the monomeric form, which is exposed on the cell surfaces of disrupted or infected cells. This may suppress excess immune responses.     

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.     

Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex

Amongst the many stimuli orienting the growth of plant roots, of critical importance are the touch signals generated as roots explore the mechanically complex soil environment. However, the molecular mechanisms behind these sensory events remain poorly defined. We report an impaired obstacle-avoiding response of roots in Arabidopsis lacking a heterotrimeric G-protein. Obstacle avoidance may utilize a touch-induced release of ATP to the extracellular space. While sequential touch stimulation revealed a strong refractory period for ATP release in response to mechano-stimulation in wild-type plants, the refractory period in mutants was attenuated, resulting in extracellular ATP accumulation. We propose that ATP acts as an extracellular signal released by mechano-stimulation and that the G-protein complex is needed for fine-tuning this response.     

Hypoxia-Inducible Factor 1 Regulation through Cross Talk between mTOR and MT1-MMP

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.     

Association between Maternal Exposure to di(2-ethylhexyl) Phthalate and Reproductive Hormone Levels in Fetal Blood: The Hokkaido Study on Environment and Children's Health

Prenatal di(2-ethylhexyl) phthalate (DEHP) exposure can produce reproductive toxicity in animal models. Only limited data exist from human studies on maternal DEHP exposure and its effects on infants. We aimed to examine the associations between DEHP exposure in utero and reproductive hormone levels in cord blood. Between 2002 and 2005, 514 pregnant women agreed to participate in the Hokkaido Study Sapporo Cohort. Maternal blood samples were taken from 23–35 weeks of gestation and the concentration of the primary metabolite of DEHP, mono(2-ethylhexyl) phthalate (MEHP), was measured. Concentrations of infant reproductive hormones including estradiol (E2), total testosterone (T), and progesterone (P4), inhibin B, insulin-like factor 3 (INSL3), steroid hormone binding globulin, follicle-stimulating hormone, and luteinizing hormone were measured from cord blood. Two hundred and two samples with both MEHP and hormones'' data were included in statistical analysis. The participants completed a self-administered questionnaire regarding information on maternal characteristics. Gestational age, birth weight and infant sex were obtained from birth records. In an adjusted linear regression analysis fit to all study participants, maternal MEHP levels were found to be associated with reduced levels of T/E2, P4, and inhibin B. For the stratified analyses for sex, inverse associations between maternal MEHP levels T/E2, P4, inhibin B, and INSL3 were statistically significant for males only. In addition, the MEHP quartile model showed a significant p-value trend for P4, inhibin B, and INSL3 decrease in males. Since inhibin B and INSL3 are major secretory products of Sertoli and Leydig cell, respectively, the results of this study suggest that DEHP exposure in utero may have adverse effects on both Sertoli and Leydig cell development in males, which agrees with the results obtained from animal studies. Comprehensive studies investigating phthalates'' exposure in humans, as well as their long-term effects on reproductive development are needed.     

Alzheimer’s disease associated AKAP9 I2558M mutation alters posttranslational modification and interactome of tau and cellular functions in CRISPR‐edited human neuronal cells

Alzheimer''s disease (AD) is a pervasive neurodegeneration disease with high heritability. In this study, we employed CRISPR‐Cas9‐engineered technology to investigate the effects of a rare mutation (rs144662445) in the A kinase anchoring protein 9 (AKAP9) gene, which is associated with AD in African Americans (AA), on tau pathology and the tau interactome in SH‐SY5Y P301L neuron‐like cells. The mutation significantly increased the level of phosphorylated tau, specifically at the site Ser396/Ser404. Moreover, analyses of the tau interactome measured by affinity purification‐mass spectrometry revealed that differentially expressed tau‐interacting proteins in AKAP9 mutant cells were associated with RNA translation, RNA localization and oxidative activity, recapitulating the tau interactome signature previously reported with human AD brain samples. Importantly, these results were further validated by functional studies showing a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared with wild type cells in a tau‐dependent manner, which are mirrored with pathological phenotype frequently seen in AD. Our results demonstrated specific effects of rs14462445 on mis‐processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.     

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.     

First application of the SINE (short interspersed repetitive element) method to infer phylogenetic relationships in reptiles: an example from the turtle superfamily Testudinoidea

Although turtles (order Testudines) constitute one of the major reptile groups, their phylogenetic relationships remain largely unresolved. Hence, we attempted to elucidate their phylogeny using the SINE (short interspersed repetitive element) method, in which the sharing of a SINE at orthologous loci is indicative of synapomorphy. First, a detailed characterization of the tortoise polIII/SINE was conducted using 10 species from eight families of hidden-necked turtles (suborder Cryptodira). Our analysis of 382 SINE sequences newly isolated in the present study revealed two subgroups, namely Cry I and Cry II, which were distinguishable according to diagnostic nucleotides in the 3' region. Furthermore, four (IA-ID) and five (IIA-IIE) different SINE types were identified within Cry I and Cry II subgroups, respectively, based on features of insertions/deletions located in the middle of the SINE sequences. The relative frequency of occurrence of the subgroups and the types of SINEs in this family were highly variable among different lineages of turtles, suggesting active differential retroposition in each lineage. Further application of the SINE method using the most retrotranspositionally active types, namely IB and IC, challenged the established phylogenetic relationships of Bataguridae and its related families. The data for 11 orthologous loci demonstrated a close relationship between Bataguridae and Testudinidae, as well as the presence of the three clades within Bataguridae. Although the SINE method has been used to infer the phylogenies of a number of vertebrate groups, it has never been applied to reptiles. The present study represents the first application of this method to a phylogenetic analysis of this class of vertebrates, and it provides detailed information on the SINE subgroups and types. This information may be applied to the phylogenetic resolution of relevant turtle lineages.