Ikuko Kadohira

Molecular Mechanisms of How Mercury Inhibits Water Permeation through Aquaporin-1: Understanding by Molecular Dynamics Simulation

Aquaporin (AQP) functions as a water-conducting pore. Mercury inhibits the water permeation through AQP. Although site-directed mutagenesis has shown that mercury binds to Cys189 during the inhibition process, it is not fully understood how this inhibits the water permeation through AQP1. We carried out 40 ns molecular dynamics simulations of bovine AQP1 tetramer with mercury (Hg-AQP1) or without mercury (Free AQP1). In Hg-AQP1, Cys191 (Cys189 in human AQP1) is converted to Cys-SHg+ in each monomer. During each last 10 ns, we observed water permeation events occurred 23 times in Free AQP1 and never in Hg-AQP1. Mercury binding did not influence the whole structure, but did induce a collapse in the orientation of several residues at the ar/R region. In Free AQP1, backbone oxygen atoms of Gly190, Cys191, and Gly192 lined, and were oriented to, the surface of the water pore channel. In Hg-AQP1, however, the SHg+ of Cys191-SHg+ was oriented toward the outside of the water pore. As a result, the backbone oxygen atoms of Gly190, Cys191, and Gly192 became disorganized and the ar/R region collapsed, thereby obstructing the permeation of water. We suggest that mercury disrupts the water pore of AQP1 through local conformational changes in the ar/R region.

Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [(32)P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

Expression and distribution of tight junction proteins in human amnion during late pregnancy.

Amnion is the innermost layer of the fetal membrane and has been suggested to regulate the volume of amniotic fluid via the amniotic epithelium. The transepithelial pathway is generally restricted by tight junctions (TJs). Thus far, human amniotic TJs have not been identified. In this study, we determined whether the human amniotic epithelium contains TJs. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting analyses showed that the human amniotic epithelium has TJ components, such as occludin, ZO-1, and at least 2 types of claudins, i.e., claudin-4 and claudin-7. The TJ components were found to localize in the lateral membranes and cytoplasm at 35 weeks of gestation; these components disappeared from the lateral membrane at 37 weeks of gestation. Organ culturing of the amnion at 37 weeks gestation induced the relocalization of the TJ proteins from the cytoplasm to the lateral membranes. Furthermore, in cultured amniotic epithelial cells, dexamethasone induced the downregulation of the protein expression of TJs. These findings suggest that the human amniotic epithelium has TJs that disrupt during late pregnancy. The disruption may be induced by several factors such as glucocorticoids present in the amniotic fluid during late pregnancy.

Comparison of the right and left ventricular performance during the fetal development using velocity vector imaging

BACKGROUND Studies on myocardial characteristics examined by speckle-tracking echocardiography are limited. AIMS To compare myocardial performance between the right and left ventricles during the fetal development using velocity vector imaging (VVI). SUBJECTS AND STUDY DESIGN Echocardiograms of 95 uncomplicated singleton fetuses (19-36 weeks pregnancy) were retrospectively analyzed by VVI to measure global longitudinal peak velocity, strain, and strain rate of both the right ventricle (RV) and left ventricle (LV). The regional values were calculated for three segments (base, mid, and apex) of the ventricular free wall and segment. OUTCOME MEASURES The VVI-derived measurements were examined for gestational age and compared between ventricles. RESULTS The global peak systolic and diastolic velocity values of both ventricles significantly increased over gestation examined, whereas the global systolic strain and strain rate were stable (RV: strain -22.6 ± 5.0%, strain rate -2.6 ± 0.7/s; LV: strain -21.5 ± 5.6%, strain rate -2.5 ± 0.7/s). Compared to the LV, the RV showed significantly higher global velocity in systole and diastole (P = 0.001 for systole, P < 0.001 for diastole). The global systolic velocity of the LV increased close to the RV toward term, whereas the RV was dominant in diastole throughout the examined gestation. Basal strain and strain rate in the RV were significantly greater than that of the LV, although there were no significant differences in the middle and apical values between ventricles. CONCLUSION Our findings suggest the RV predominance of longitudinal contraction and dilatation, compared to the LV in uncomplicated fetuses.

Prenatal Images of the Aortopulmonary Window With an Interrupted Aortic Arch

A 35-year-old woman, gravid 2 para 1, was referred to our clinic for detailed sonographic evaluation at 29 weeks gestation because of fetal growth restriction. Fetal echocardiography demonstrated a situs solitus with normal atrioventricular and ventriculoarterial connections. However, the left and right ventricular outflow tracts were combined just above the semilunar valves, representing a k-shaped vascular complex in the three-vessel trachea (3VT) view (Fig. 1a, Movie 1). Color Doppler sonography showed the communication of the arterial blood flow between the ascending aorta and the pulmonary artery, indicating an aortopulmonary window (APW) (Fig. 1b, Movie 1). The shunt flow was in a right-to-left direction. The ascending aorta appeared to be hypoplastic without tracing into the descending aorta. The brachiocephalic artery and the left carotid artery originated from the ascending aorta, whereas the left subclavian artery was supplied by the ductus arteriosus. The estimated body weight of the fetus was in the 15th percentile for gestational age of Japanese fetuses. The diagnosis determined for the fetus was APW with type B interrupted aortic arch (IAA). At 38 weeks gestation, a male infant weighing 2,100 g was delivered by cesarean section due to his breech presentation. His Apgar scores were seven at 1 min and nine at 5 min. The prenatal diagnosis was confirmed by both postnatal three-dimensional computed tomography and echocardiography (Fig. 2). Direct closure of APW with reconstruction of the aortic arch was performed 5 days after the neonate’s birth. The APW, defined as a defect between the ascending aorta and the pulmonary artery above the normal semilunar valves, is a rare heart disease accounting for 0.1–0.2 % of all cardiac anomalies in neonates [1]. Patients with isolated APW could develop progressive heart failure caused by high pulmonary blood flow [2]. Additionally, nearly half of patients with APW show other cardiac lesions, including conotruncal abnormalities and septal defects [1]. Among conotruncal abnormalities, type A interruption is seen frequently in APW [1]. In this combination of APW with IAA, IAA seems to be an independent risk for mortality, although the survival rate of the patients is high after optimal intervention [3]. Therefore, prenatal detection of APW in combination with IAA could contribute to early surgical treatments for the improvement of mortality and morbidity [4]. In the reported case, a k-shaped vascular complex in the 3-VT view was the distinctive finding of APW in combination with IAA. To date, case reports on prenatal findings of APW are limited [2, 5]. Hayashi et al. [5] described a Y-shaped confluence of the ascending aorta and the pulmonary artery indicating APW with IAA, which is substantially consistent with our findings. Taken together, the APW could be diagnosed in the 3VT view, giving clinicians a clue to detect other cardiac lesions. Especially the kor Y-shaped vascular complex in the 3VT view should evoke APW with IAA for appropriate treatment immediately after birth. Electronic supplementary material The online version of this article (doi:10.1007/s00246-012-0613-0) contains supplementary material, which is available to authorized users.

Complete chorion-amnion separation presenting as a stuck fetus

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