Mechanism of Vitamin D Receptor Ligand-Binding Domain Regulation Studied by gREST Simulations.

The vitamin D receptor ligand-binding domain (VDR-LBD) undergoes conformational changes upon ligand binding. In this nuclear receptor family, agonistic or antagonistic activities are controlled by the conformation of the helix (H)12. However, all crystal structures of VDR-LBD reported to date correspond to the active H12 conformation, regardless of agonist/antagonist binding. To understand the mechanism of VDR-LBD regulation structurally, conformational samplings of agonist- and antagonist-bound rat VDR-LBD were performed using the generalized replica exchange with solute tempering (gREST) method. The gREST simulations demonstrated different structural responses of rat VDR-LBD to agonist or antagonist binding, whereas in conventional molecular dynamics simulations, the conformation was the same as that of the crystal structures, regardless of agonist/antagonist binding. In the gREST simulations, a spontaneous conformational change of H12 was observed only for the antagonist complex. The different responses to agonist/antagonist binding were attributed to hydrophobic core formation at the ligand-binding pocket and cooperative rearrangements of H11. The gREST method can be applied to the examination of structure-activity relationships for multiple VDR-LBD ligands.

Isolation of Jahanene and Jahanane, and Total Synthesis of the Jahanyne Family.

Two new jahanyne analogues, jahanene and jahanane, highly N-methylated lipopeptides, were isolated from a marine cyanobacterium Okeania sp., and their structures were determined by NMR and MS. In addition, we achieved total syntheses of the jahanyne family and assessed their activities. The resulting growth-inhibitory activity of jahanyne was nearly one-tenth of the previously reported activity. Furthermore, we found that the degree of unsaturation at the terminus of the fatty acid moiety affected the growth-inhibitory activity against human cancer cells.

A-kinase anchoring protein 3 messenger RNA expression in ovarian cancer and its implication on prognosis.

A-kinase anchoring protein 3 (AKAP3) is a sperm protein and its expression appears to be restricted to the testis in normal adult tissues. We investigated AKAP3 mRNA expression in 20 normal ovaries and 54 ovarian cancers of different histological types, grades and stages by reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were analyzed by conventional agarose gel electrophoresis and capillary electrophoresis on a microtip device to determine the expression semiquantitatively. Little or no expression was observed in the 20 normal ovarian specimens. High AKAP3 mRNA expression was observed in 15 ovarian cancer specimens (28 %). The expression was correlated with the histological grade and clinical stage. AKAP3 mRNA was observed at a significantly higher frequency in poorly differentiated (p = 0.009) and advanced stage (III and IV, p = 0.014) tumors. No correlation was found between AKAP3 mRNA expression and other variables. In Cox multivariate analysis, AKAP3 mRNA expression was found to be a significant predictor of both overall and progression-free survival in patients with poorly differentiated tumors.

Expression and potential roles of pregnane X receptor in endometrial cancer.

Estrogen has been shown to contribute greatly to growth and development in endometrial cancer. And recent research has suggested that intratumoral production of estrogen may play important roles in this cancer tissue. On the other hand, pregnane X receptor (PXR), a new member of nuclear receptors, has been shown to mediate the genomic effects of steroid hormones, including estrogen and xenobiotics. And this receptor is thought to regulate the expression of the cytochrome P-450 3A (CYP3A) gene family, which plays important roles in the metabolism of endogenous steroids and xenobiotics. Various levels of PXR expression were found in endometrial cancer tissues but not normal tissues. Tissues showing high PXR expression showed significantly high expression of CYP3A4/7 and low expression of estrogen receptor (ER) compared with levels in tissues showing low PXR expression. In endometrial cancer cell lines, HEC-1 cells, which express high PXR and low ER and progesterone receptor, show a stronger transcriptional response of the PXR-CYP3A pathway to the PXR ligands, especially endocrine-disrupting chemical, than do Ishikawa cells. These data suggest that the steroid/xenobiotics metabolism in the tumor tissue through PXR-CYP3A pathway might play an important role, especially in alternative pathway for gonadal hormone and endocrine-disrupting chemical effects on endometrial cancer expressing low ER alpha.

Nafamostat mesilate suppresses NF-kappaB activation and NO overproduction in LPS-treated macrophages.

Nafamostat mesilate (NM), a clinically used serine protease inhibitor, suppressed the overproduction of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) in RAW264.7 murine macrophages treated with lipopolysaccharide (LPS, 100 ng/ml); however, it had little effect on endothelial NOS (eNOS) in human umbilical vein endothelial cells (HUVEC). Electrophoretic mobility shift assay (EMSA) revealed that LPS activated nuclear factor-kappaB (NF-kappaB) in RAW264.7 cells and that this activation was suppressed by nafamostat mesilate. Western blotting showed that nafamostat mesilate suppressed the phosphorylation and degradation of inhibitor kappaB-alpha (IkappaB-alpha), which holds NF-kappaB in the cytoplasm in an inactivated state. Our observations suggest that nafamostat mesilate is a candidate agent for various diseases such as ischemia-reperfusion, graft rejection, inflammatory diseases, and autoimmune diseases, in which iNOS and/or NF-kappaB are upregulated.

Increased plasma adrenomedullin in women with recurrent pregnancy loss.

OBJECTIVE: To evaluate vascular changes and uterine perfusion in women with recurrent pregnancy loss. METHODS: We measured plasma levels of adrenomedullin of 100 pregnant women in the midluteal phase of a nonpregnant cycle (control group: n = 62; recurrent pregnancy loss group: n = 38). We measured the pulsatility index (PI) in the uterine arteries by transvaginal pulsed Doppler ultrasonography at the same time. RESULTS: The plasma level of adrenomedullin in women with recurrent pregnancy loss (5.6 +/- 1.9, mean +/- standard deviation) was significantly higher (P >.001) than that in control women (3.6 +/- 1.7). Uterine arterial PI of women with recurrent pregnancy loss (2.70 +/- 0.47) was significantly higher (P >.001) than that in control women (2.09 +/- 0.39). Plasma level of adrenomedullin had a significant positive correlation with uterine arterial PI both in the control group (r =.58, P <.001) and in the recurrent pregnancy loss group (r =.78, P <.001). Both plasma adrenomedullin concentration (7.2 +/- 2.3) and uterine arterial PI (3.06 +/- 0.36) were significantly high in women with antiphospholipid antibodies. CONCLUSION: Plasma adrenomedullin may serve as a useful biochemical marker for recurrent pregnancy loss caused by impaired uterine perfusion.

The relationship between Doppler indices from inferior vena cava and hepatic veins in normal human fetuses.

This study was conducted to determine the gestational age-related reference range of the preload index [peak velocity during atrial contraction (A)/peak velocity during ventricular systole (S)] for the inferior vena cava (IVC), the right hepatic vein, the middle hepatic vein and the left hepatic vein. The slope and the intercept of the regression line for each preload index were compared among the 4 veins using analysis of covariance. Doppler measurements were obtained for the 4 veins of 316 normal fetuses at 22-40 weeks of gestation. A and S values were measured from the recorded flow velocity waveform of each vein and the A/S ratio was calculated as the preload index. The regression lines for the preload index of the 4 veins decreased gradually throughout gestation. Analysis of covariance revealed no significant differences in the slopes of the regression lines for the 4 veins. However, the intercepts of the regression lines for all hepatic veins were significantly higher than that of the regression line for the IVC (P<0.0001), with the difference ranging from 0.024 to 0.033. There were no significant differences among the intercepts of the regression lines for different hepatic veins. We concluded that the relationship between the preload index and the duration of gestation was statistically similar for all hepatic veins, and strongly resembled that for the IVC.

Prediction markers for respiratory distress syndrome: evaluation of the stable microbubble test, surfactant protein-A and hepatocyte growth factor levels in amniotic fluid.

Surfactant treatment in infants with respiratory distress syndrome (RDS) has decreased neonatal mortality. With the advent of this therapy, it has become important to predict accurately the fetal lung maturity of a fetus before delivery. We evaluated the stable microbubble test (SMT), surfactant protein-A (SP-A) and hepatocyte growth factor (HGF) in amniotic fluid as predicting markers for RDS. Of 55 amniotic fluid samples obtained by amniocentesis from women less than 37 weeks pregnant, the SMT values were as follows: sensitivity 76.5%, specificity 84.2%, positive predictive value 68.4%, negative predictive value 88.9% and overall accuracy 81.8%. For SP-A, the values were 88.2%, 65.8%, 53.6%, 92.6% and 72.7%, respectively. If we used both SMT and SP-A, we could diagnose with 100% accuracy that a case with measurements of SMT > or = 2 and SP-A > or = 420 ng/ml would not complicate with RDS (24/24). However, the RDS diagnostic accuracy of HGF does not equal to those of SMT and SP-A levels. We concluded that the rapidity, simplicity and reliability of SMT was very useful during 24-36 weeks of gestation as a bedside procedure to predict fetuses likely to develop RDS. We also noted the additive effect of SP-A in improving the accuracy of lung maturity diagnosis.

Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts.

OBJECTIVE: To examine whether or not peroxynitrite was involved in trophoblastic apoptosis induced by a bacterial endotoxin, lipopolysaccharide (LPS). METHODS: Levels of nitrite/nitrate, stable metabolites of nitric oxide (NO), in culture medium of trophoblasts, were determined using Griess reagents. Trophoblastic apoptosis was identified morphologically and confirmed using in situ nick end labeling technique. The amount of nitrotyrosine, a footprint of peroxynitrite, was quantified by dot blotting. Statistical significance was determined by ANOVA. RESULTS: Treatment of trophoblasts with LPS leads to apoptosis accompanied by formation of NO and nitrotyrosine. Aminoguanidine, an inhibitor of NO synthase (NOS), reduced peroxynitrite formation and prevented apoptosis. Scavengers of peroxynitrite also prevented apoptosis in this culture model. CONCLUSION: Peroxynitrite was involved in trophoblastic apoptosis induced by LPS. Peroxynitrite scavengers or inhibitors of NOS may thus be candidate therapeutic agents for infectious diseases, which is associated with overproduction of NO and peroxynitrite.

[Paclitaxel and carboplatin with or without pirarubicin (THP-ADR) as first line chemotherapy in elderly patients].

To evaluate the validity of administration of paclitaxel and carboplatin with or without pirarubicin (THP-ADR) as first line chemotherapy in elderly patients with gynecologic cancer, we explored the efficacy and safety of these regimens. From October 1, 1998 to September 30, 2001, we administered paclitaxel and carboplatin with or without THP-ADR pursuant to the chart we prepared originally as first line chemotherapy in patients with gynecologic cancer. Eleven elderly patients (age > 70 years) and 62 younger patients (age < 70 years) were entered into the present study. Paclitaxel was administered as a 3-hour intravenous (i.v.) infusion at dosages of 135 to 180 mg/m2 immediately followed by carboplatin over 60 minutes at dosages of area under the curve (AUC) 3 to 5, administered intravenously or intraperitoneally. We observed grade 3/4 anemia more frequently in elderly patients receiving the regimen including paclitaxel and carboplatin without THP-ADR (9% v.s. 47%, p < 0.0001). Grade 3/4 anemia (10% v.s. 22%, p = 0.02) and grade 3/4 thrombocytopenia (7% v.s. 22%, p = 0.007), febrile neutropenia (14% v.s. 44%, p = 0.02) also occurred more frequently in elderly patients receiving the regimen including paclitaxel and carboplatin with THP-ADR. The overall response rates were equivalent among elderly and younger patients (69% and 78%), respectively. The regimen consisting of paclitaxel and carboplatin without THP-ADR was applied safely to elderly patients.

Impaired uterine arterial blood flow in pregnant women with recurrent pregnancy loss.

OBJECTIVE: This study was undertaken to evaluate uterine perfusion, which regulates uterine receptivity, in women with recurrent pregnancy loss. METHODS: We evaluated the blood flow resistance in the uterine arteries of 104 pregnant women at 4 to 5 weeks' gestation by transvaginal pulsed Doppler ultrasonography (control group, n = 52; and recurrent pregnancy loss group, n = 52). Blood tests for antinuclear and antiphospholipid antibodies were also performed. RESULTS: The uterine arterial pulsatility index in the recurrent pregnancy loss group was significantly higher than that in the control group. Women with antinuclear or antiphospholipid antibodies had an elevated pulsatility index in the uterine artery, which is prominent in women with recurrent pregnancy loss. Coagulopathy and vascular dysfunction caused by autoantibodies may impair uterine perfusion. However, the uterine arterial pulsatility index in the recurrent pregnancy loss group was significantly higher than that in the control group even among women without antinuclear antibodies or among women without antiphospholipid antibodies. This observation strongly suggests that the uterine artery pulsatility index may be an independent index for recurrent pregnancy loss. CONCLUSIONS: The introduction of pulsed Doppler ultrasonography has provided the means for noninvasive evaluation of uterine impedance and may identify patients with recurrent pregnancy loss associated with impaired uterine perfusion.

Changes in the aortic and pulmonary diameters in a fetus with hypoplastic left heart syndrome.

We diagnosed hypoplastic left heart syndrome in a 26-week-old fetus using fetal echocardiography. Color Doppler ultrasonography was helpful for evaluating the structural abnormalities. The diameters of the aorta and the pulmonary artery were measured periodically from 26 to 38 weeks of gestation. Aortic diameter was below the normal range throughout gestation. The diameter of the pulmonary artery was normal at 26 weeks of gestation but gradually dilated and was abnormally dilated after the 36th week of gestation. Here we discuss the cause of enlarged pulmonary artery in fetal hypoplastic left heart syndrome.

Cervical carcinoma with full-thickness stromal invasion: efficacy of dynamic MR imaging in the assessment of parametrial involvement.

The purpose of this study was to investigate the efficacy of dynamic MR imaging in the assessment of parametrial involvement by cervical carcinoma with full-thickness stromal invasion on thin-section oblique axial T2-weighted images. Dynamic MR images of 24 patients with cervical carcinoma with full-thickness stromal invasion on thin-section oblique axial T2-weighted images were evaluated with pathologic correlation. Dynamic MR imaging was performed using a turboFLASH, 3D-FISP, or 2D-FLASH technique. The imaging planes of dynamic MR imaging were oblique axial planes of the uterine cervix. Dynamic MR imaging was performed twice, once for the early phase (40 to 60 sec after the administration of contrast media) and once for the late phase (5 min). Contrast enhancement of the tumor was divided into six types. Type I, cervical stroma with low signal intensity surrounding a tumor with high signal intensity, was seen in the early phase of dynamic MR imaging; type II-RR, the hyperintense rim was seen from the early phase to the late phase; type II-RO, the hyperintense rim was seen in the early phase only; type II-OR, the hyperintense rim was seen in the late phase only; type II-O, the hyperintense rim was not seen at all; and type III, tumor invasion with high signal intensities was seen beyond the cervical stroma in the early phase of dynamic MR imaging. The numbers for each type of cervical carcinoma on dynamic MR images were as follows: type I, four parametrial sites; type II-RR, 0; type II-RO, 0; type II-OR, 13; type II-O, 14; and type III, one. Three-dimensional diameters (transverse, craniocaudal, and anteroposterior) of the primary tumor were measured using dividers. All parametrial sites of type I and type II-OR showed no parametrial involvement. One parametrial site of type III and three parametrial sites of type II-O showed parametrial involvement, and 11 of type II-O showed no parametrial involvement. None of the patients showed type I-RR or type II-RO. When type I and type II-OR were categorized as criteria of no parametrial involvement and type III and transverse diameters of 3.5 cm or over classified as type II-O were categorized as criteria of parametrial involvement, the rate of diagnostic accuracy was 95.8%. Dynamic MR imaging is considered to be substantially useful in the assessment of parametrial involvement with cervical carcinoma with full-thickness stromal invasion by thin-section oblique axial T2-weighted images.

Differential blood flow in uterine, ophthalmic, and brachial arteries of preeclamptic women.

OBJECTIVE: To develop a method that employs noninvasive, pulsed Doppler ultrasonography combined with measurement of flow-mediated vasodilation to evaluate characteristic endothelial dysfunction in various degrees of preeclampsia. METHODS: Uterine, ophthalmic, and brachial arterial blood flow of 99 pregnant women (control group [n = 32], non-preeclamptic intrauterine growth restriction group (n = 15), mild preeclampsia group [n = 25], and severe preeclampsia group [n = 27]) were evaluated by pulsed Doppler ultrasound or flow-mediated vasodilation. RESULTS: Uterine, orbital, and brachial circulation were altered in preeclampsia, whereas no significant differences were observed between the non-preeclamptic intrauterine growth restriction and control groups. Pulsatility index in the uterine arteries of preeclamptic women with intrauterine growth restriction was approximately three-fold higher than that of normotensive women with or without intrauterine growth restriction. The peak ratio (defined to quantify characteristic flow velocity waveform) of the ophthalmic artery of hypertensive women was significantly higher than that of normotensive women. Flow-mediated vasodilation in the brachial artery of preeclamptic women with intrauterine growth restriction was significantly lower than that in preeclamptic women without intrauterine growth restriction. Among preeclamptic women, elevation of the resistance in the uterine artery and reduced flow-mediated vasodilation were closely correlated to intrauterine growth restriction, whereas the elevated peak ratio of the ophthalmic artery was dependent on hypertension, irrespective of the presence of intrauterine growth restriction. CONCLUSION: Ultrasound evaluation of uterine and orbital circulation and flow-mediated vasodilation of the brachial artery helps differentiate the degree and severity of preeclampsia.

Bisphenol A stimulates NO synthesis through a non-genomic estrogen receptor-mediated mechanism in mouse endothelial cells.

Biological actions of bisphenol A (BPA), an environmental chemical, have not been fully elucidated. We studied effect of BPA on nitric oxide (NO) synthesis in the murine endothelial cell line, MSS31. BPA (1-100 microM) increased nitrite/nitrate, a stable metabolites of NO, levels in culture medium of MSS31. However, Western blotting showed that the level of endothelial NO synthase protein was not increased by 16 h of treatment with BPA (10 microM). ICI 182,780 (10 microM), an estrogen receptor (ER) antagonist, suppressed BPA-induced NO synthesis while actinomycin D (1 microg/ml), a transcription inhibitor, or cycloheximide (40 microM), a protein synthesis inhibitor, exhibited no effect on BPA-induced NO synthesis. These results indicate that BPA stimulates NO synthesis through a non-genomic ER-mediated mechanism. Short-term effects of BPA on NO synthesis were weak but similar to 17beta-estradiol.

A long-term transdermal nitric oxide donor improves uteroplacental circulation in women with preeclampsia.

OBJECTIVE: To determine the effects of long-term transdermal administration (range, 4-30 days; mean +/- SD, 11.1+/-7.2 days) of isosorbide dinitrate, a nitric oxide donor, in preeclamptic women. METHODS: We studied uterine and fetoplacental circulation of 12 preeclamptic women with oligohydramnios and an elevated pulsatility index in the uterine arteries. RESULTS: Transdermal isosorbide dinitrate significantly suppressed the blood pressure of patients. Pulsed Doppler ultrasonography revealed that the average pulsatility index in the uterine arteries was significantly reduced by treatment with isosorbide dinitrate (P < .003). The average pulsatility index in the umbilical artery was also significantly reduced (P < .004). Furthermore, the size of the amniotic fluid pocket increased approximately 4-fold by treatment with isosorbide dinitrate. CONCLUSIONS: Long-term transdermal administration of isosorbide dinitrate improves fetoplacental circulation and may be effective therapy for avoiding maternal hypertension and oligohydramnios in some preeclamptic women.

The biological effects of antiadhesion agents on activated RAW264.7 macrophages.

The objective of this study is to determine the biological effects of various antiadhesion agents on macrophages, which play an essential role in wound healing and adhesion. To determine these effects, RAW264.7 macrophages were activated with lipopolysaccharide in the presence of antiadhesion agents: oxidized regenerated cellulose (oxyC), sodium hyaluronate (HA), dexamethasone (Dex), or chondroitin sulfate (CS). The release of nitric oxide (NO), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), or matrix metalloproteinases (MMPs) from RAW264.7 was measured. We found that oxyC reduced the release of NO, IL-6, MMP-2, and MMP-9, whereas it enhanced the release of VEGF. HA reduced the release of MMP-2, whereas it enhanced the release of VEGF and NO. HA exhibited no significant effect on the release of IL-6 or MMP-9. Dex reduced the release of NO, VEGF, IL-6, MMP-2, and MMP-9. CS reduced the release of VEGF, IL-6, and MMP-2, although it had no significant effect on the release of NO and MMP-9. Antiadhesion agents, which have been clinically used as physical barriers, modulated the functions of macrophages.

[The effect of L-Glutamine and Shakuyaku-Kanzo-to for paclitaxel-induced myalgia/arthralgia].

Myalgia/arthralgia is a crucial side effect of paclitaxel, and may become the major dose-limiting side effect. However, this is a situation where there is little effective preventive treatment. L-Glutamine was reported as a neuroprotective agent for vincristine-induced neurotoxicity. In Japan, there have been reports on steroid and Shakuyaku-Kanzou-to (a herbal medicine) for paclitaxel-induced myalgia/arthralgia. This study aimed to compare the effect of L-Glutamine and Shakuyaku-Kanzou-to, and to discuss the validity of these agents for the paclitaxel-induced myalgia/arthralgia. Our results suggested that Shakuyaku-Kanzou-to showed no remarkable effects against paclitaxel-induced myalgia/arthralgia as had been reported before; however, both L-Glutamine and Shakuyaku-Kanzou-to decreased the duration of grade 2 toxicity (CALGB Expanded Common Toxicity Criteria) in comparison with those who were not treated. L-Glutamine and Shakuyaku-Kanzou-to might therefore a preventive effect against moderate or severer myalgia/arthralgia during paclitaxel-treated chemotherapy. Further trials are needed to confirm the value of these drugs.

Effect of a nitric oxide donor on the ophthalmic artery flow velocity waveform in preeclamptic women.

OBJECTIVE: To evaluate the effects of an antihypertensive agent on the orbital circulation of preeclamptic women. METHODS: We studied the ophthalmic arteries of 10 healthy pregnant women and 10 women with severe preeclampsia by pulsed Doppler ultrasonography and evaluated the effect of transdermal isosorbide dinitrate, a nitric oxide donor, on preeclamptic women. RESULTS: The average pulsatility index and resistive index were significantly lower, whereas the average end-diastolic velocity, time-averaged mean peak velocity, and peak ratio, which quantifies characteristic changes in the ophthalmic artery flow velocity waveform, were higher in preeclamptic women. Transdermal isosorbide dinitrate significantly reduced the average end-diastolic velocity (P < .05) and peak ratio of the ophthalmic artery (P < .01), whereas it did not significantly affect other indices. CONCLUSIONS: Orbital circulation was altered in preeclamptic women. A nitric oxide donor affected orbital circulation. Peak ratio was a sensitive index for evaluating orbital circulation in preeclampsia.