Contribution of fetal ANXA5 gene promoter polymorphisms to the onset of pre-eclampsia.

OBJECTIVE: A common haplotype M2 consisting of minor SNP alleles located in the ANXA5 gene promoter region has been described as a risk factor for various obstetric complications such as recurrent pregnancy loss, pre-eclampsia and pregnancy-related thrombophilic disorder. However, the question of whether it is the maternal or fetal genotype that contributes to the onset of these disorders remains to be resolved. METHODS: We analyzed ANXA5 gene variants in the blood and placental tissues from pre-eclampsia patients and normotensive controls. ANXA5 expression was examined by qRT-PCR, Western blotting and immunostaining. Results were compared between M2 and non-M2 carriers. RESULTS: The M2 haplotype was found to be significantly frequent in placentas from pre-eclamptic patients relative to the controls (25.5% versus 10%, P = 0.044), In contrast, no significant differences were observed in maternal blood (13.0% versus 11.3%, P = 0.597). The placental expression of ANXA5 mRNA was found to be lower in M2 carriers. When examined by Western blot and immunostaining, the ANXA5 protein levels were found to be affected more by the placental than the maternal genotype. Histological examination of the placentas from the pre-eclamptic patients demonstrated that a placental M2 haplotype correlated more closely than maternal M2 with the severity of perivillous fibrin deposition. CONCLUSIONS: Although preliminary, these results suggest that hypomorphic M2 alleles in the in placental ANXA5 promoter, whether transmitted maternally or paternally, might be an essential determinant of an increased risk of pre-eclampsia via local thrombophilia at the feto-maternal interface.

Female pseudohermaphroditism associated with maternal steroid cell tumor, not otherwise specified of the ovary: a case report and literature review.

Maternal virilization in pregnancy with or without fetal female pseudohermaphroditism has several etiologies. Of these, pregnancy luteoma is the most common cause of maternal virilization during pregnancy, and approximately 20 cases have been reported in recent years. Moreover, four cases of pregnancy luteomas with female pseudohermaphroditism have been reported. However, the extremely rare steroid cell tumor, not otherwise specified (NOS), has been reported only once as a cause for maternal virilization. Herein, the authors report the first case of maternal virilization with female pseudohermaphroditism associated with steroid cell tumor-NOS along with the clinical course, pathological features, and a review of the literature.

Upregulation of HtrA4 in the placentas of patients with severe pre-eclampsia.

High temperature requirement A (HtrA) family proteins are serine proteases that may serve in the quality control of misfolded or mislocalized proteins. Recently, possible involvements of HtrA1 in the normal development of the placenta and in the pathogenesis of pre-eclampsia were reported. In this study, we characterized HtrA4, a previously uncharacterized HtrA protein family member, in pre-eclampsia. Elevated expression levels of placental HtrA4 in pre-eclampsia patients were observed by qRT-PCR. Western blotting also showed an increased production of HtrA4 at the protein level in pre-eclamptic placentas. In normal chorionic villi, HtrA4 protein was more abundant in the cytoplasm of cytotrophoblasts than in syncytiotrophoblasts. In contrast, the amount of HtrA4 protein in syncytiotrophoblasts was dramatically increased in pre-eclamptic placentas. Circulating HtrA4 was detected at higher levels in sera from women with pre-eclampsia than from those with normotensive pregnancies. Serum HtrA4 levels were higher in patients with early onset and inversely correlated with the weights of the newborn and placenta. Furthermore, serum levels correlated with serum PAPP-A and PAPP-A2 levels, indicating a functional role for HtrA4 in the common pathway. These data suggest that increased HtrA4 may be involved in the onset of pre-eclampsia, and elevated levels in sera imply a potential application as a biomarker for this disorder.

A transgenic mouse model for investigating the response of the upstream region of whey acidic protein (WAP) gene to various steroid hormones.

The limitations of studies of clarification of response elements of whey acidic protein (WAP) gene to hormones using mammary cell lines has been shown. We studied the response of the upstream region (2.6 kb) of WAP to various steroid hormones using gonadectomized mWAP/hGH transgenic mice. Ovariectomy or castration for transgenic mice was performed at 10 days or 30 days post partum. Various steroid hormones were administered daily for 10 days to the gonadectomized transgenic mice after they reached 2 months of age. Prior to the hormonal administration and 24 hr after the final administration, blood was collected and the hGH levels in the plasma was measured by RIA. Daily doses of estradiol-17 beta were significantly more effective at increasing hGH levels in transgenic females ovariectomized at 10 days post partum than progesterone of an equal dose. A combined dose of progesterone and of estradiol-17 beta significantly amplified the increase of hGH levels accompanied by the great development of mammary glands, compared to a dose of progesterone alone. Corticosterone induced only a slight increase of hGH, while testosterone had no effect. The doses of gonadal steroid hormones did not induce an increase in hGH levels and development of mammary glands in the castrated transgenic males. The results showed that the response of 5' region of WAP requires at least some extended development of the mammary gland and that the 2.6 kb upstream region of the exogenous WAP gene contained the element responsive to ovarian hormones.

Hunting the subset-specific genes of neuroblastoma: expression profiling and differential screening of the full-length-enriched oligo-capping cDNA libraries.

BACKGROUND: Neuroblastoma (NBL) has a distinct nature in different prognostic subgroups. PROCEDURE: To understand the molecular mechanism of NBL's genesis and biology as well as that of the neural crest development, we constructed full-length-enriched cDNA libraries by an oligo-capping method from two different subsets of primary NBL, one with favorable biology and the other with MYCN amplification. RESULTS: Sequencing analysis of these libraries revealed that the expression profile was markedly different between both subsets. To identify the genes differentially expressed between the subsets, semi-quantitative RT-PCR analyses are proceeding. CONCLUSION: So far, 54 transcripts have been found to be expressed at high levels in favorable NBLs, and significantly at low levels in unfavorable NBLs.

Lipoprotein(a), left atrial appendage function and thromboembolic risk in patients with chronic nonvalvular atrial fibrillation.

Lipoprotein(a) (Lp(a)) has a prothrombotic effect by modulating the fibrinolytic system. The purpose of the present study was to determine whether serum Lp(a) levels are associated with an increased risk of thromboembolism in chronic nonvalvular atrial fibrillation (NVAF). Clinical, laboratory and transesophageal echocardiographic data were collected in 172 consecutive, non-anticoagulated patients with chronic NVAF. Thirty-four patients (thromboembolic group) had a recent (<1 month) embolic event and/or a left atrial thrombus on transesophageal echocardiography. The thromboembolic group had a higher frequency of spontaneous echo contrast (94 vs. 58%, p<0.0001), increased concentrations of Lp(a) (median: 31.5 vs. 15.5 mg/dl, p<0.0001) and fibrinogen (median: 352 vs. 314 mg/dl, p = 0.0015), larger left atrial dimensions (median: 5.1 vs. 4.8cm, p = 0.0078), and reduced left atrial appendage (LAA) flow velocities (median: 9.5 vs. 21.2 cm/s, p<0.0001) than the nonthromboembolic group. Multivariate analysis identified 3 independent predictors of thromboembolism: Lp(a) level > or =30 mg/dl (odds ratio (OR) 9.5, 95% confidence interval (CI) 4.4-20.4, p<0.0001), LAA flow velocity of <20 cm/s (OR 8.7, 95% CI 3.3-23.0, p = 0.0003) and a fibrinogen concentration of <377mg/dl (OR 3.2, 95% CI 1.5-6.9, p = 0.0201). The Lp(a) elevations and reduced LAA flow velocities are independently associated with thromboembolism in chronic NVAF.

Differential regulation of immediate early gene expression in preadipocyte cells through multiple signaling pathways.

Using digoxigenin (DIG)-based differential hybridization, a series of immediate early genes (IEG) was identified following the adipogenic stimulation in 3T3-L1 preadipocyte cells. Most of the known IEGs were identified as well as new members such as zf9 and Stra13. To delineate possible signaling pathways accounting for these gene expression, a subset of specific kinase inhibitors, SB203580, PD98059, rapamycin, LY294002, and Ro-32-0432, which inhibit p38 (HOG), MEK (MAPKK), S6 kinase, PI3 kinase, and protein kinase C (PKC), respectively, were employed. The IEGs were classified into three categories according to their susceptibility to the inhibitors. Expression of the first group (c-fos, jun-B, egr-1, tis11, tis21, thrombospondin-1, erp, thyroid hormone receptor [N-10], cyr61, and zf9) was mainly dependent on PKC and MEK pathways, while that of the second class (gene33 and tis10) exhibited an additional dependence on PI3 kinase pathways. The third one (Id-3, gly96, and Stra13) was characterized in that none of these inhibitors interfered with gene expression. Our results suggest that the induction of IEGs by the adipogenic stimuli is mediated by common as well as distinct signaling pathways.

cDNA cloning and expression analysis of mouse zf9, a Krüppel-like transcription factor gene that is induced by adipogenic hormonal stimulation in 3T3-L1 cells.

Using a differential hybridization method, we have cloned a zinc finger transcription factor gene whose expression was enhanced by adipogenic hormones in preadipocyte 3T3-L1 cells. Cloning of this gene revealed that it encodes a mouse homologue of rat Zf9 and human CPBP/GBF, previously identified as a wound-induced transcription factor and GC-rich binding protein, respectively. The mRNA for this clone consisted of 0.9 kb coding region and 3.2 kb long 3' untranslated region. Northern blot analysis revealed that it was ubiquitously expressed, among adult tissues, in which abundant expression was observed in lung, ovary and thymus. The transcript was transiently induced by different stimuli such as serum, cAMP and 12-O-tetradecanoylphorbol 13-acetate. Nuclear run-on and RNA synthesis inhibitor chase experiments indicated that the transient induction of the mRNA was regulated both at transcriptional and post-transcriptional levels.

Expression of milk protein genes is induced directly by exogenous STAT5 without prolactin-mediated signal transduction in transgenic mice.

STAT5 is a member of the family of STAT (signal transducer for activating transcription), which was isolated from nuclear extracts of the lactating gland of sheep. It was reported that, in vitro, STAT5 could be activated directly by prolactin (PRL) without PRL-mediated signal transduction. The present study was conducted to investigate above the possibility in vivo, using the transgenic mice overexpressing ovine STAT5 (oSTAT5) under the control of MMTV promoter. Three transgenic mouse lines that expressed the exogenously introduced oSTAT5 in their mammary glands were established. The expression levels of exogenous oSTAT5 were higher than those of endogenous STAT in the mammary glands of all of the three transgenic lines. Although the expression levels of endogenous milk protein genes, WAP, and beta-casein genes, were not correlated with oSTAT5 expression, the expression levels of WAP and beta-casein were induced by exogenous oSTAT5 in the transgenic mice. The present study demonstrated, at very least, that STAT5 expression can directly activate the expression of milk protein genes, and particularly the WAP gene without PRL-mediated signal transduction.

Elevated serum lipoprotein(a) is a risk factor for left atrial thrombus in patients with chronic atrial fibrillation: a transesophageal echocardiographic study.

BACKGROUND: Patients with chronic atrial fibrillation have an increased risk of thromboembolism. Apoprotein(a) has a structural homology with plasminogen, suggesting that lipoprotein(a) [Lp(a)] may produce thrombogenic effects by modulating the fibrinolytic system. However, the role of Lp(a) level in the formation of left atrial thrombus has not been studied. We sought to evaluate whether Lp(a) is a risk factor for left atrial thrombus in patients with chronic atrial fibrillation. METHODS AND RESULTS: The consecutive series of 150 patients (mean age 67 +/- 8 years) with chronic atrial fibrillation underwent transesophageal echocardiography. Left atrial thrombus was diagnosed by transesophageal echocardiography. Clinical, biochemical, and echocardiographic variables were prospectively collected. Univariate analysis showed that patients with left atrial thrombus (n = 29, 19%) had higher frequency of spontaneous echo contrast (93% vs 55%, P <.0001) than patients without left atrial thrombus (n = 121). Patients with left atrial thrombus also had a significantly higher serum concentration of Lp(a) (34.5 +/- 24.1 vs 17.9 +/- 13.5 mg/dL, P <.0001), a larger left atrium (5.4 +/- 0.9 vs 4.8 +/- 0.7 cm, P <.001), and a lower left atrial appendage peak flow velocity (11.1 +/- 5.4 vs 23.5 +/- 14.6 cm/s, P <.0001). Multivariate regression analysis showed that the Lp(a) concentration (P <.0001) was a significant positive predictor and the left atrial appendage peak flow velocity (P =.0125) was a significant negative predictor of left atrial thrombus. Left atrial thrombus was present in 16 (48%) of 33 patients with Lp(a) level >/=30 mg/dL. CONCLUSIONS: Elevated serum levels of Lp(a) are strongly associated with left atrial thrombus. These findings suggest that Lp(a) level may be a novel risk factor for left atrial thrombus in patients with chronic atrial fibrillation.

Saphenous vein graft shrinkage as a mechanism of stenosis soon after bypass surgery.

A 63-year-old man with left main coronary artery disease underwent aortocoronary bypass surgery using saphenous vein grafts. Less than 1 month later, severe narrowing occurred in the mid-portion of the vein graft to the left anterior descending coronary artery. Preintervention intravascular ultrasonography revealed prominent vein graft shrinkage. Percutaneous transluminal angioplasty failed because the stenotic lesion could not be dilated, even by high-pressure balloon inflation. Saphenous vein graft shrinkage appears to be one of the mechanisms of early saphenous vein graft stenosis, and balloon angioplasty to the vein graft stenosis with prominent shrinkage may be of only limited value.

A sensitive ELISA for the characterization of two forms of circulating intercellular adhesion molecule-1 in human plasma.

Nine murine monoclonal antibodies (MAbs) raised against recombinant soluble intercellular adhesion molecule-1 (sICAM-1) were evaluated by means of ELISAs, and their neutralizing activity was investigated in two bioassays employing Raji cells activated with phorbol myristate acetate. Three of the MAbs inhibited autoaggregation of the activated cells and adhesion to sICAM-1 fixed on a plastic plate. Non-neutralizing antibody SM1-255 is directed to an epitope that was not recognized by the other eight MAbs. This property enabled us to develop two ELISAs employing SM1-255 as a liquid-phase antibody for the quantitation of sICAM-1 circulating (cICAM-1) in human plasma. One assay, employing non-neutralizing antibody WIS2-11 as a solid-phase, has a sensitivity of two pg/well with coefficients of variation of 3.6-5.8% (within assay) and 5.5-9.5% (between assay). The other assay, employing neutralizing antibody WIS5-85 as a solid-phase, has a sensitivity of four pg/well with coefficients of variation of 2.7-7.2% (within assay) and 9.2-11.2% (between assay). There was a discrepancy between cICAM-1 levels of human plasma determined by these two assays. All samples showed 2- to 5-times higher levels in the assay using WIS2-11 than in that using WIS5-85. The result from the gel electrophoresis employing Western blotting suggests that ICAM-1 circulates in at least two molecular forms with molecular masses of about 85 and 130 kDa and with different reactivities to WIS2-11 and WIS5-85.

Nicorandil suppresses a hump on the monophasic action potential and torsade de pointes in a patient with idiopathic long QT syndrome.

The patient was a 71-year-old female with Torsade de Pointes (TdP) associated with idiopathic long QT syndrome. TdP and polymorphic nonsustained VT were frequently observed at bedside and an electrophysiologic study was performed. The QT (and QTU) interval was abnormally prolonged, and alteration of the QT interval was also recorded on the electrocardiogram. Monophasic action potential (MAP) from the right ventricle showed a hump on the falling limb of the MAP following a long RR interval of more than 1.0 sec. Intravenous administration of nicorandil (2 mg) resulted in disappearance of the hump, and ventricular arrhythmia was no longer observed. The QT interval at a PP interval of 720 msec was slightly shortened. She was treated with a DDD-pacemaker and given nicorandil. No recurrence of TdP was observed during the follow-up period of 8 months. This drug might be effective in patients with idiopathic long QT syndrome.

Sudden cardiac death from hypertrophic cardiomyopathy and acute idiopathic (Fiedler's) myocarditis: autopsy report.

A 45-year-old man with a rapidly deteriorating heart condition died suddenly, two hours after admission, after resistance to attempts of cardiac pacing. At autopsy, the heart weighed 600g with asymmetric septal hypertrophy. Histological examination revealed an extensive and diffuse disarray of myocardial fibers in the ventricular septum and in the free wall of both ventricles. Pronounced mononuclear cell infiltrations and interstitial edema were distributed widely in both ventricles. There were few abnormal findings in the other organs. The diagnosis was hypertrophic cardiomyopathy accompanied by Fiedler's myocarditis. Such a case appears to be rare.

The effects of m-octopamine on salivary flow rates and protein secretion by rat submandibular glands.

1. m-Octopamine given i.v. or i.p. was a potent sialogogue for rat salivary glands. 2. Salivation in response to i.v. m-octopamine was completely abolished by prazosin and phenoxybenzamine. 3. The alpha-type of proteins were secreted in response to all doses of i.v. and i.p. m-octopamine and these were converted into the beta-type with prazosin, but not with yohimbine. 4. m-Octopamine stimulated both alpha- and beta-adrenoceptors and was a much more selective alpha 1-agonist than was the p-isomer.

Restricted expression of N- and R-cadherin on neurites of the developing chicken CNS.

The expression of two cadherins, N- and R-cadherin, was mapped in the CNS of chicken embryos of 6-11 d incubation, focusing on the sensory and motor fiber systems. In the spinal cord, the laterally located fibers of the dorsal funiculus express N-cadherin while the medially located fibers do not. These two fiber systems have a different course within the CNS but associate to form the spinal dorsal roots. In the hindbrain, N-cadherin is expressed by the descending trigeminal (general somatic sensory) tract, which is contiguous with the N-cadherin-positive zone of the dorsal funiculus of the spinal cord. R-cadherin is not expressed by sensory fibers, but is expressed by the visceral motor system of the vagus and glossopharyngeal nerves, which are N-cadherin negative. The motor neurites expressing R-cadherin have a different course within the brain than the sensory neurites expressing N-cadherin, although they form the common sensory/motor roots of the vagus nerve at the surface of the brain. The possibility that N-cadherin provides a guidance cue for sensory axon migration within the CNS by a homophilic adhesion mechanism was investigated in vitro. Explants from sensory spinal ganglia expressing N-cadherin were placed on N-cadherin-transfected neuroblastoma cells, and axon outgrowth was visualized. Results showed that the sensory axons defasciculate and closely follow the cell-cell boundaries between transfected cells where high levels of N-cadherin are expressed. These results show that the two cadherins, like members of the immunoglobulin superfamily of molecules, are expressed in a topographically restricted fashion during chick brain development. They furthermore suggest that N-cadherin expression by neurites may play a role in guiding these neurites along CNS paths that express the same molecule.

[Morphological and functional studies on implanted arterial graft].

Early morphological and functional changes in arterial graft were studied. Autologous free graft of dog femoral artery or vein was implanted in the arterial system and the changes of endothelial cell by scanning electron microscope and prostacyclin levels were observed. In arterial graft, endothelial cell detachment after the implantation was rarely observed except the site with mechanical damage around the anastomosis. The site of cell detachment regenerated by 21 days after the implantation. The prostacyclin level was 2.5 pg/mg in venous graft or 35.9 pg/mg in arterial graft. The level in venous graft decreased after the implantation and it increased according to the cell regeneration, while the level was stable in arterial graft. The prostacyclin level of internal thoracic artery or gastroepiploic artery in clinical use was 54.5 pg/mg or 50.1 pg/mg, respectively. However the level in saphenous vein (SV) was 17.3 pg/mg and there were significant differences between the levels of arterial grafts and one of SV. Conclusively the arterial graft was superior to venous graft in early morphological and functional changes after the implantation and the superiority in early period after graft implantation may be one of the factor to contribute good graft patency in long term period.

Morphological and functional study of free arterial grafts.

Morphological and functional changes of free arterial grafts in dogs were studied for 3 weeks after implantation and the changes were compared to those in implanted free vein grafts. In the arterial grafts, endothelial cells with abundant pinocytotic vesicles and some cytoplasmic folds were observed by transmission and scanning electron microscope and cell detachment was seen only at the site of anastomosis, while most cells were detached in the vein grafts. The site of mechanical damage in the arterial grafts was covered by regenerated endothelial cells which showed similar morphological findings to the normal arterial endothelial cells. In contrast, regenerated cells in the vein grafts started to cover the denuded area 7 days after the implantation and had completely covered it by 3 weeks. Prostacyclin was produced more abundantly in arterial grafts than in vein grafts at any phase after implantation. The level of prostacyclin production was between 30 and 40 pg/mg in any phase after implantation of free arterial grafts, while in vein grafts the level was 2.5 pg/mg at the day of implantation and increased to 13.6 pg/mg at 21 days. This study showed that the endothelial cells were well preserved and the level of prostacyclin production was high in the arterial grafts, and thus the grafts seemed to show potent anti-thrombogenicity after implantation. Although late changes in arterial and vein grafts were not investigated in this experimental protocol, these results may suggest that the arterial graft is superior to the vein graft even in the early period after its implantation as a free graft.