Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury.

Maternal inflammation is associated with spontaneous preterm birth and respiratory impairment among premature infants. Recently, molecular hydrogen (H2) has been reported to have a suppressive effect on oxidative stress and inflammation. The aim of this study was to evaluate the effects of H2 on fetal lung injury caused by maternal inflammation. Cell viability and the production of interleukin-6 (IL-6) and reactive oxygen species (ROS) were examined by treatment with lipopolysaccharide (LPS) contained in ordinal or H2-rich medium (HM) using a human lung epithelial cell line, A549. Pregnant Sprague Dawley rats were divided into three groups: Control, LPS, and HW + LPS groups. Rats were injected with phosphate-buffered saline (Control) or LPS intraperitoneally (LPS) on gestational day 19 and provided H2 water (HW) ad libitum for 24 h before LPS injection (HW + LPS). Fetal lung samples were collected on day 20, and the levels of apoptosis, oxidative damage, IL-6, and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry. The number of apoptotic cells, and levels of ROS and IL-6 were significantly increased by LPS treatment, and repressed following cultured with HM in A549 cells. In the rat models, the population positive for cleaved caspase-3, 8-hydroxy-2'-deoxyguanosine, IL-6, and VEGF was significantly increased in the LPS group compared with that observed in the Control group and significantly decreased in the HW + LPS group. In this study, LPS administration induced apoptosis and oxidative damage in fetal lung cells that was ameliorated by maternal H2 intake. Antenatal H2 administration may decrease the pulmonary mobility associated with inflammation in premature infants.

Association study of genetic variants on chromosome 7q31 with susceptibility to normal tension glaucoma in a Japanese population.

The caveolin 1 to caveolin 2 (CAV1-CAV2) gene region on chromosome 7q31 has been reported to be associated with susceptibility to primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) in previous studies. We investigated whether genetic variants in the CAV1-CAV2 region are associated with NTG in Japanese patients. Two hundred and ninety-two Japanese patients with NTG and 352 Japanese healthy controls were recruited. We genotyped three single-nucleotide polymorphisms; that is, rs1052990, rs4236601, and rs7795356, in the CAV1-CAV2 gene region and assessed the allelic diversity among cases and controls. The frequency of the minor allele (G) of rs1052990 was significantly decreased in NTG cases compared with controls (P=0.014, OR=0.71), whereas NTG or POAG cases had a significantly higher frequency of the allele than controls in previous studies. Conversely, rs7795356 did not show any significant association with NTG cases, and rs4236601 was monomorphic in the Japanese study population. Our findings did not correspond with previous positive results, suggesting that CAV1-CAV2 variants studied in the present study are not important risk factors for NTG susceptibility in all populations. Further studies are needed to elucidate the possible contribution of the CAV1-CAV2 region to the development of glaucoma.

Magnetic Compton scattering studies of magneto-dielectric Ba(Co0.85Mn0.15)O3-δ

We revealed that the Ba(Co0.85Mn0.15)O3-δ ceramic samples exhibited ferromagnetic-dielectric behavior below the magnetic transition temperature of about 35 K. The origin of their magnetic ordering was expected to super-exchange coupling of Co4+(d5)-O2--Mn4+(d3) with bonding angle of 180° and/or Mn4+(d3)-O2--Mn4+(d3) with bonding angle of 90°. The magnetic spin momentum estimated by the magnetic Compton profiles (MCP) of the samples had similar temperature dependence as that determined by the temperature dependence of magnetic moment by superconducting quantum interference device, which meant that the observed magnetic moments could be ascribed to the spin moment. The shapes of the MCPs of the samples were completely same regardless of the temperature measured. This result indicates that there are no changes of the momentum space distribution of spin density between ferromagnetic and paramagnetic states. So, this magnetic transition is simply caused by a thermal fluctuation of the spin.

Increased expression of sphingosine kinase in the amnion during labor.

INTRODUCTION: Sphingosine-1-phosphate (S1P), a bioactive lipid, has been reported to regulate inflammation processes. The onset of labor is thought to be related to inflammation. We therefore hypothesized that S1P might be involved in the onset of labor. METHODS: The expression of sphingosine kinase (SPHK)-1, which produces S1P, and S1P lyase (SPL)-1, which irreversibly inactivates S1P, were examined in the fetal membranes. The expression levels were compared between amnions from cases of elective Caesarean deliveries (pre-labor) and those from vaginal deliveries (post-labor). In primary cultured human amnion cells, the expression levels of prostaglandin-endoperoxide synthase (PTGS)-2 were examined in the presence or absence of S1P treatment. RESULTS: SPHK-1 and SPL-1 were both expressed in the amnion. The expression of SPHK-1 in the post-labor amnions increased compared with that in the pre-labor amnions. The expression of PTGS-2, a key regulator of labor, also increased in the post-labor amnion. However, the SPL-1 expression in the pre-labor amnion was not significantly different from that in the post-labor amnion. S1P1-3 and 5, which were coupled with Gi protein, were consistently found in the amnion cells. The treatment with S1P increased the expression of PTGS-2, and this was completely suppressed by a Gi inhibitor in the amnion cells. DISCUSSION: We are herein provide the first evidence of increased SPHK-1 expression in post-labor amnions, and that S1P increases the PTGS-2 expression in amnion cells. CONCLUSIONS: Our results suggest that S1P might play a role in the onset of labor via the induction of PTGS-2.

A case report of placental mesenchymal dysplasia with an increased VEGF-D expression.

The pathogenesis of placental mesenchymal dysplasia (PMD) remains unclear. This report presents a case of PMD with a female fetus complicated with intrauterine growth restriction (IUGR). The ultrasound findings were similar to molar pregnancies, but PMD was suspected based on the presence of low ß-hCG levels and a normal karyotype. After delivery, pathological examination of the placenta showed dilated villi and thick-walled vessels lacking trophoblast proliferation, which thus led to a diagnosis of PMD. The VEGF-D (Xp22.31) mRNA expression was found to have increased in the abnormal villi. Whether this is an incidental or X-linked gene specific event in, IUGR complicated, PMD pathogenesis warrants further investigation of VEGF-D expression in PMD.

MFM and PEEM observation of micrometre-sized magnetic dot arrays fabricated by ion-microbeam irradiation in FeRh thin films.

FeRh thin films were irradiated with a 10 MeV iodine ion microbeam to produce micrometre-scale ferromagnetic microstructures by modifying the local magnetic character from antiferromagnetism to ferromagnetism using ion-microbeam irradiation. Two-dimensional magnetic dot arrays of dimensions ∼2 µm × 4 µm as well as 10 µm × 10 µm were successfully produced on the FeRh surface, which was confirmed by magnetic force microscopy (MFM). The results of photoemission electron microscopy (PEEM) combined with X-ray magnetic circular dichroism reveal that the easy axis of the magnetization of the ion-beam-irradiated ferromagnetism in the FeRh thin films lies in the film planes along the <001> direction of the MgO substrates.

Psychological stress during in vitro fertilization and embryo transfer is influenced by the patients' background and gender.

Purpose: This study evaluated the changes in psychological stress during in vitro fertilization and embryo transfer (IVF-ET) and the relationship of such stress to the patients' background and gender. Methods: Sixty couples undergoing IVF-ET were administered the State-Trait Anxiety Inventory-JYZ (STAI) test at six different points during IVF-ET procedures. Anxiety scores at each time point were recorded and analyzed according to gender, fertility status, and duration of treatment. Results: The median state anxiety score for women increased following induction until oocyte collection, after which it temporarily declined and then increased again until the pregnancy test. No such changes were noted in men. Scores for women who had undergone a shorter period of IVF treatments were higher while state and trait anxiety in men increased with a prolonged treatment period. Unsuccessful treatment increased the state and trait anxiety of women. Conclusions: Psychological stress changed periodically depending on the duration of the patients' treatment and fertility status also influenced anxiety levels. These findings will prove helpful in guiding psychological therapy and counseling for couples attempting to conceive by in vitro fertilization.

Investigation of the association between the GLC3A locus and normal tension glaucoma in Japanese patients by microsatellite analysis.

PURPOSE: To investigate whether the GLC3A locus harboring the CYP1B1 gene is associated with normal tension glaucoma (NTG) in Japanese patients. MATERIALS AND METHODS: One hundred forty-two Japanese patients with NTG and 101 Japanese healthy controls were recruited. Patients exhibiting a comparatively early onset were selected as this suggests that genetic factors may show stronger involvement. Genotyping and assessment of allelic diversity was performed on 13 highly polymorphic microsatellite markers in and around the GLC3A locus. RESULTS: There were decreased frequencies of the 444 allele of D2S0416i and the 258 allele of D2S0425i in cases compared to controls (P = 0.022 and P = 0.034, respectively). However, this statistical significance disappeared when corrected (Pc > 0.05). We did not find any significant association between the remaining 11 microsatellite markers, including D2S177, which may be associated with CYP1B1, and NTG (P > 0.05). CONCLUSIONS: Our study showed no association between the GLCA3 locus and NTG, suggesting that the CYP1B1 gene, which is reportedly involved in a range of glaucoma phenotypes, may not be an associated factor in the pathogenesis of NTG.

Population-based prevalence of optic disc haemorrhages in elderly Japanese.

PURPOSE: To estimate the population-based prevalence of disc haemorrhages (DHs) in an elderly Japanese population and evaluate related factors including optic disc morphology. PATIENTS AND METHODS: Colour fundus photographs were taken during the screening examination of the Tajimi Study, during which 3021 of 3870 eligible residents of the city aged 40 years or older were screened (response rate, 78.1%). All fundus photographs were graded by one masked examiner to determine the presence of DHs. RESULTS: Good quality fundus photographs were available for both eyes of 2761 subjects. DHs were found in at least one eye of 34 subjects (1.2%; 95% confidence interval (CI) 0.8-1.6%). The prevalence was 14.0% (95% CI, 8.0-19.9%), 9.7% (2.9-16.6%), and 0.4% (0.1-0.6%) in subjects with definitive glaucoma, glaucoma suspects, and subjects without glaucoma, respectively. Logistic regression analyses indicated that DHs were associated with glaucoma (P<0.001), glaucoma suspects (P<0.001), and older age (P=0.032). No Heidelberg Retina Tomograph parameters differed between subjects with and without DHs. CONCLUSIONS: The prevalence of DHs was 1.2% in an elderly Japanese population, which was similar to other population studies. In addition to glaucoma, older age was associated with higher prevalence of DHs.

Ocular and systemic factors related to intraocular pressure in Japanese adults: the Tajimi study.

BACKGROUND: As intraocular pressure (IOP) and age are consistent risk factors of glaucoma, it is of special interest to know the association between IOP and possibly relating factors including age in Japan where a high prevalence of normal-tension glaucoma has been reported. The aim of this report was to evaluate the distribution of and factors related to applanation IOP in a population-based study in Japan. METHODS: A randomly sampled group of 3021 residents (response rate 78.1%) of Tajimi City, aged 40 years or older, underwent screening examinations including measurements of IOP with Goldmann applanation tonometry and central corneal thickness. RESULTS: Among right eyes without glaucoma, suspected glaucoma or other disorders which could affect correct IOP measurements, IOP averaged 14.6 (SD 2.7) and 14.5 (2.5) mm Hg in men and women, respectively, with no significant intergender difference (p = 0.342). Multiple regression analyses revealed that age was significantly negatively correlated with IOP (non-standardised beta (B) = -0.020/year, p = 0.0001). Higher body mass index (B = 0.14/BMI, p<0.0001), higher mean blood pressure (B = 0.022/mm Hg, p<0.0001), history of diabetes (p = 0.0019), thicker cornea (B = 0.014/microm, p<0.0001), higher myopia (B = 0.055/dioptres, p = 0.0043) and steeper corneal curvature (B = -0.72/mm, p = 0.0002) were also significantly correlated with higher IOP. CONCLUSIONS: In an adult Japanese population, applanation IOP averaged 14.5 mm Hg and was negatively correlated with age after adjusting for other related factors. A positive correlation between IOP and myopia was found.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase.

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

Angiotensin II augmented migration and invasion of choriocarcinoma cells involves PI3K activation through the AT1 receptor.

While angiotensin II (Ang II) has been shown to inhibit migration of extravillous trophoblasts via plasminogen activator inhibitor-1 (PAI-1) activation, it has remained unclear whether it stimulates or inhibits malignant behavior of choriocarcinoma cells. Since we previously found an involvement of the renin-angiotensin system (RAS) in the proliferative potential in choriocarcinoma cells (BeWo), mediated via the Ang II type 1 receptor (AT1R), in the present study we investigated the effects of Ang II on choriocarcinoma cell migration/invasion in vitro using Transwell cell culture chambers. Ang II (10(-8)M) promoted migration and invasion by a choriocarcinoma cell line and augmented random cell mobility on checkerboard analysis. Immunoblotting showed Ang II to activate the phosphorylation of FAK and Akt in BeWo cells. Furthermore Ang II effects on cell migration were abolished by a selective AT1R antagonist and a phosphatidylinositol 3-kinase (PI3K) inhibitor. The present results suggest that Ang II-induced migration and invasion of choriocarcinoma cells probably involves PI3K following binding to the AT1R.

Oxytocin-induced prostaglandin E2 (PGE2) synthesis is regulated by progesterone via oxytocinase in Ishikawa cells.

Cell-surface oxytocinase inactivates oxytocin and regulates oxytocin stimulation. We reported that oxytocinase in human endometrial epithelial cells was secreted from the cell membrane in the mid-secretory phase and disappeared from the cell surface. On the other hand, the production in human endometrium of prostaglandins, which play important roles in the reproductive process, has been reported to be upregulated by oxytocin. We investigated whether progesterone affects cell-surface oxytocinase and oxytocin-induced prostaglandin E2 (PGE2) production in vitro. Progesterone induced secretion of oxytocinase into the culture medium, which resulted in a decrease in cell-surface oxytocinase. Production of PGE2 was increased slightly by oxytocin without progesterone, and significantly with progesterone. The inhibition of oxytocinase activity by amastatin had a similar effect to the loss of cell-surface oxytocinase caused by progesterone. It is therefore likely that the cell-surface oxytocinase of endometrial epithelial cells modified by progesterone plays an important role in the function of the human endometrium through PGE2.

Usefulness of peripheral anterior chamber depth assessment in glaucoma screening.

PURPOSE: This study was conducted to determine the usefulness of peripheral anterior chamber depth assessment in angle-closure glaucoma (ACG) screening in Japanese subjects. SUBJECTS AND METHODS: The subjects were 14,779 adults 40 years old or older. Eyes having peripheral anterior chamber depth that is 1/4 the peripheral corneal thickness (van Herick's classification: grade 2) and less than 1/4 the peripheral corneal thickness (van Herick's classification: grade 1) were extracted as narrow angle eyes, and those eyes were further examined. RESULTS: Of 14,779 subjects, 923 eyes of 505 subjects were diagnosed as narrow angle eyes (3.4%). Narrow angle eyes were observed in 4.9% of female subjects and 1.9% of male subjects, indicating a significantly higher frequency in women. The percentage of narrow angle eyes increased with age. Among the narrow angle eyes, 61 eyes of 32 subjects were diagnosed with ACG suspect (6.5%). In contrast to the frequency of ACG suspect in eyes classified as grade 1, according to van Herick's classification, being 17.9%, that in eyes classified as grade 2 was significantly lower at 5.6%. CONCLUSION: Since the incidence of ACG suspect increases as the peripheral anterior chamber depth decreases, caution for the peripheral anterior chamber depth is required for the ACG screening.

Frequency doubling technology and confocal scanning ophthalmoscopic optic disc analysis in open-angle glaucoma with hemifield defects.

PURPOSE: To evaluate the potential of frequency doubling technology for detecting early glaucomatous damage. PATIENTS AND METHODS: Forty-nine eyes of 49 patients with open-angle glaucoma with visual field defects only in one hemifield according to the Humphrey Field Analyzer 30-2 program were included. Forty-five healthy patients were also included as control subjects. In each patient, frequency doubling technology with the threshold N-30 program and optic disc analysis using the Heidelberg Retina Tomograph was performed. Frequency doubling technology test results and the Humphrey Field Analyzer test results were compared. Optic disc parameters corresponding to the hemifield designated intact by the Humphrey Field Analyzer were compared between the eyes in which the hemifield was normal by frequency doubling technology and those in which the hemifield was abnormal. RESULTS: Forty-one percent of the 49 hemifields designated intact by the Humphrey Field Analyzer were abnormal based on frequency doubling technology, whereas 98% of the 49 hemifields designated defective by the Humphrey Field Analyzer were abnormal and 12% of the 90 hemifields designated intact by the Humphrey Field Analyzer were abnormal in healthy patients. The percentage of the hemifields designated abnormal by frequency doubling technology was significantly higher than that in healthy patients (P < 0.001). The rim volume was significantly smaller in eyes with abnormal results based on frequency doubling technology than in eyes with normal results (P < 0.05, paired t test, with Bonferroni correction for multiple comparison). CONCLUSION: Frequency doubling technology can detect glaucomatous damage earlier than conventional static perimetry can.

Characterization of a secretase activity for placental leucine aminopeptidase.

Placental leucine aminopeptidase (P-LAP) is believed to play an important role in the inactivation of small regulatory peptides. P-LAP exists in both membrane-bound and soluble forms and cDNA cloning has demonstrated that P-LAP is a type II membrane protein, which means that its soluble form is released by a specific proteolytic cleavage. In this report, we studied this process in COS7 cells. Inhibitors of serine or aspartic proteases did not affect the secretion of P-LAP, while EDTA and 1,10-phenanthroline inhibited it. In addition, we transfected P-LAP expression vectors that have point mutations of the cleavage site or deletion of the juxtamembrane stalk. Point mutations of the cleavage site resulted in significantly lower secretion of P-LAP. On the contrary, the distance to cleavage site showed no relation to P-LAP secretion. These results suggest that P-LAP secretase has a metalloprotease activity which depends on the amino acid sequence of the cleavage site.

Transcriptional regulation of human placental leucine aminopeptidase/oxytocinase gene.

Human placental leucine aminopeptidase (P-LAP) plays a major role in the clearance of oxytocin, which is a key hormone in regulating labour pain. To explore the transcriptional regulation of P-LAP gene expression in placenta, we performed systematic studies using human choriocarcinoma cells, BeWo and JEG-3, as a model of placental trophoblastic cells. Transient transfection and luciferase assays using various 5'-deleted P-LAP-luciferase constructs showed that the region from -297 to +49 of the transcription start site was responsible for promoter activity in these cells. Footprinting analysis with nuclear extracts from both cell lines demonstrated at least four sites for nucleoprotein interactions in this region (FP1 to FP4). Site-directed deletion of FP1-4 in luciferase assays indicated the significance of the FP3 region (-214 to -183) for high promoter activity in the cells. Electrophoretic mobility shift assays to identify the proteins interacting with DNA at FP3 revealed three retarded bands, one of which was generated by activator protein-2 (AP-2) binding. Our findings suggest that AP-2 may be one of the important factors regulating P-LAP gene expression in human placenta.

Optimal doses of paclitaxel and carboplatin combination chemotherapy for ovarian cancer: a phase I modified continual reassessment method study.

BACKGROUND: A multicenter, phase I study of combination therapy with paclitaxel and carboplatin for epithelial ovarian cancer was conducted to determine the safety and recommended dosages for Japanese women. METHODS: Paclitaxel was administered intravenously over a 3-h period, followed by carboplatin administered intravenously over a 1.5-h period. A modified continual reassessment method (mCRM) was used in two treatment arms to establish the maximum tolerated dose (MTD) and recommended doses of the combination. In group A, the dose of paclitaxel (175 mg/m2) was constant and the dose of carboplatin was increased from 4 to 7 in terms of the target area under the plasma concentration-versus-time curve (AUC). In group B, the dose of carboplatin was constant (AUC 6) and paclitaxel was administered at two dose levels (160 and 175 mg/m2). In both groups, the carboplatin dose was limited to a maximum of 800 mg/body for each administration. RESULTS: Because the calculated probability of toxicity was greatest at a dose of paclitaxel 175 mg/m2 and carboplatin AUC 7, this dose was designated the MTD in group A. Based on this result, treatment in group B was initiated at doses of paclitaxel of 160 mg/m2 and carboplatin AUC 6. While the dose of paclitaxel was escalated to 175 mg/m2, the safety of the combination was confirmed. The most frequent adverse effect was neutropenia, which resolved promptly with the appropriate use of granulocyte-colony stimulating factor (G-CSF). No other severe hematologic or nonhematologic toxicities were observed. CONCLUSIONS: Our study demonstrated that the recommended dose for this combination regimen should be paclitaxel 175 mg/m2 plus carboplatin AUC 6 (maximum dose, 800 mg/body).

Oxytocin stimulates the translocation of oxytocinase of human vascular endothelial cells via activation of oxytocin receptors.

Oxytocinase (OTase) degrades several small peptides such as oxytocin (OT), and thus plays important roles in fetal development and maintenance of human homeostasis during pregnancy. The physiological effects of OT are mediated via its receptor (OTR). Although the interactions between OT and OTR have studied extensively, the relationship to OTase remains to be clarified. It is known that human umbilical vascular endothelial cells express OTR messenger RNA; therefore, they were selected for examination of this question in the present study. RT-PCR experiments confirmed the existence of messenger RNA for OTase, and assessment of protein levels and activity clarified that OT increases the activity of OTase at the cell surface via binding to OTR. This stimulation appears to involve translocation of OTase from cytosolic to the cell surface in response to cellular signal transduction pathways linked to the OTR. Protein kinase C stimulation significantly increased the cell surface activity of OTase, whereas its inhibition resulted in reduction. In summary, our findings provide clear evidence that OT triggers directly OTase translocation in human umbilical vascular endothelial cells via a protein kinase C-dependent pathway coupled to OTR.