p63(TP63) elicits strong trans-activation of the MFG-E8/lactadherin/BA46 gene through interactions between the TA and DeltaN isoforms.

We report here that human MFGE8 encoding milk fat globule-EGF factor 8 protein (MFG-E8), also termed 46 kDa breast epithelial antigen and lactadherin, is transcriptionally activated by p63, or TP63, a p53 (TP53) family protein frequently overexpressed in head-and-neck squamous cell carcinomas, mammary carcinomas and so on. Despite that human MFG-E8 was originally identified as a breast cancer marker, and has recently been reported to provide peptides for cancer immunotherapy, its transcriptional control remains an open question. Observations in immunohistochemical analyses, a tetracycline-induced p63 expression system and keratinocyte cultures suggested a physiological link between p63 and MFGE8. By reporter assays with immediately upstream regions of MFGE8, we determined that the trans-activator (TA) isoforms of p63 activate MFGE8 transcription though a p53/p63 motif at -370, which was confirmed by a chromatin immunoprecipitation experiment. Upon siRNA-mediated p63 silencing in a squamous cell carcinoma line, MFG-E8 production decreased to diminish Saos-2 cell adhesion. Interestingly, the DeltaN-p63 isoform lacking the TA domain enhanced the MFGE8-activating function of TA-p63, if DeltaN-p63 was dominant over TA-p63 as typically observed in undifferentiated keratinocytes and squamous cell carcinomas, implying a self-regulatory mechanism of p63 by the TA:DeltaN association. MFG-E8 may provide a novel pathway of epithelial-nonepithelial cell interactions inducible by p63, probably in pathological processes.

Analysis of molecular interactions of the p53-family p51(p63) gene products in a yeast two-hybrid system: homotypic and heterotypic interactions and association with p53-regulatory factors.

p51 in the p53 tumor suppressor family, also referred to as p63, encodes multiple isoforms including p51A (TAp63gamma) and p51B (TAp63alpha). The p53 protein forms a tetramer, and its stability and activity are regulated by molecular association with viral and cellular proteins and by biochemical modifications. Using a yeast two-hybrid system, the p51A and p51B isoforms were examined for homotypic and heterotypic interactions in the p53 family proteins and for their affinity to the p53-regulatory factors. Results indicate a homotypic interaction dependent on the presumed oligomerization domain of the p51 proteins. The possibility of a weak heterotypic interaction between p51 and p73 proteins was suggested, while association between p51 and p53 appeared improbable. Furthermore, unlike p53, the p51 proteins failed to display an affinity to SV40 large T antigen or MDM2-family proteins. Having several features in common with p53, the p51 proteins may function in biological processes apart from p53.

So-called mesothelial/monocytic incidental cardiac excrescences obtained during valve replacement surgery: report of three cases and literature review.

We present three cases of so-called mesothelial/monocytic incidental cardiac excrescences (MICE) of the heart and a brief review of related literature. Case 1 was a 51-year-old woman who underwent mitral- and aortic-valve replacement. A tissue sample was submitted as a thrombus attached to the left atrial endocardium. Case 2 was a 69-year-old woman who underwent mitral-valve replacement. The sample was incidentally obtained as whitish clot-like fragments, but its exact origin was not known. Case 3 was a 68-year-old woman who underwent mitral-valve replacement for suspected infective endocarditis. The sample adherent to the pericardium was removed after valvular surgery. Histologically, these lesions were composed of a mixture of plump histiocytoid cells, a papillary arrangement of cuboidal cells, various sized vacuoles, and fibrin. The nests of cuboidal cells resembled cancer cells but showed features of mesothelial cells and no proliferative activity, immunohistochemically or ultrastructurally. In all cases, a suction tube placed in the left atrium was occasionally used to remove overflowing intrapericardial fluid during the surgery. The tip of the suction tube was covered with spiral wire, which is likely to transfer the stripped pericardial mesothelial cells to the left atrium. The significance of MICE is their possibility of being misdiagnosed as metastatic carcinoma by pathologists and a risk of arterial embolization by mesothelial debris clinically.

Viability of cryopreserved semilunar valves: an evaluation of cytosolic and mitochondrial activities.

BACKGROUND: Despite long-standing, widespread use of cryopreserved allografts, the basic cellular biology of these tissues is still yet unknown. The present investigation was undertaken to study cryopreserved heart valves from the standpoint of cytosolic esterase and mitochondrial dehydrogenase activities. METHODS: Cryopreserved porcine aortic cusps were observed in an unfixed fresh condition with a confocal laser scanning microscope using fluorescent dye. Porcine cusps and cultured human umbilical vein endothelial cells were divided into three groups, including fresh, cold-preserved, and cryopreserved specimens, and cytosolic esterase activity and mitochondrial dehydrogenase activity were analyzed in each. RESULTS: Confocal laser scanning microscope findings disclosed a widely distributed fluorescence in the cusp. Cytosolic esterase activity within human umbilical vein endothelial cells (28%+/-9.0%) after cryopreservation was significantly less than that it was in the cusps (72%+/-21%). Mitochondrial dehydrogenase activity of cryopreserved human umbilical vein endothelial cells and that of cusps fell to 44%+/-6.1% and 64%+/-17% respectively; the difference between the two values was not significant. CONCLUSIONS: Cryopreservation appeared to produce serious damage to cytosolic and mitochondrial functions of endothelial cells. The cytosolic function of cusps, mainly consisting of fibroblasts, was comparatively preserved after cryopreservation, but mitochondrial function of the cusps was more diminished.

p51A (TAp63gamma), a p53 homolog, accumulates in response to DNA damage for cell regulation.

p51A, or TAp63gamma, a translation product of gene p51, or p63, was identified as a homolog of p53 in its primary structure and transactivating function. p53 plays a decision-making role in inducing either cell cycle arrest or apoptosis in response to DNA damage, and thereby preserves genome integrity of living cells. To compare the biological activities between p51A and p53, cell lines with low-level, constitutive expression of each protein were obtained by cDNA transfection of mouse erythroleukemic cells. Production of p51A with an apparent molecular mass of 57-kilodalton (kD) accompanied induction of p21waf1 and appearance of hemoglobin-producing cells. After DNA-damaging treatment either with ultraviolet light (UV) irradiation or with actinomycin D, the p51A protein accumulated in time courses corresponding to those of wild-type p53, and caused an increase in the hemoglobin-positive cell count. In contrast, p53-accumulated cells underwent apoptosis without exhibiting the feature of erythroid differentiation. The mode of p21waf1 and Bax-alpha upregulations varied between p51A- and p53-expressing cells and between the types of DNA damage. These results suggest the possibility that p51A induces differentiation under genotoxic circumstances. There may be cellular factors that control p51A protein stability and transactivating ability.

Direct intraoperative measurements of aortic and pulmonary blood flows in patients with severe pulmonary artery hypertension.

BACKGROUND: Evaluate the significance of direct intraoperative measurements of aortic and pulmonary blood flows by electromagnetic flowmeter as an absolute decision basis for operability in patients with ventricular septal defect/complete atrioventricular septal defect and severe pulmonary artery hypertension. EXPERIMENTAL DESIGN: Prospective study. SETTING: Institutional practice. PATIENTS: Eight patients with marginal operability based on preoperative Doppler echocardiography and cardiac catheterization (pulmonary-to-systemic flow ratio=1.1-2.3, pulmonary-to-systemic resistance ratio=0.34-0.91, and pulmonary vascular resistance=4.6-18.2 units x m2) underwent direct intraoperative measurements of aortic and pulmonary blood flows by electromagnetic flowmeter. Operation would be performed according to the results of direct intraoperative measurements in every patient. RESULTS: Aortic flow by direct intraoperative measurements ranged from 0.9 to 3.2 L/min/m2, and pulmonary blood flow from 4.1 to 8.4 L/min/m2. Pulmonary-to-aortic flow ratio was calculated at 2.1-6.6. Pulmonary vascular resistance ranged from 2.6 to 7.7 units x m2. We assessed that all patients still had operability, and performed corrective operations. Postoperative courses corresponded with the data from the direct intraoperative measurements. CONCLUSIONS: When some clinical findings, particularly Doppler echocardiographic findings, of these patients are slightly in favor of reversibility of pulmonary vascular disease despite discrepant data of preoperative cardiac catheterization under a tight control of carbon dioxide tension, we recommend that direct intraoperative measurement of aortic and pulmonary blood flows is especially useful in decision making for the operability of patients with severe pulmonary artery hypertension.

Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a purified enzyme.

Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat enzyme with a hexahistidine tag in a baculovirus-insect cell expression system, and purified the enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 micromol/min/mg protein. The purified recombinant enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the purified enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4x10(-3) (37 degrees C, pH 9.0). These experimental results with a purified enzyme preparation quantitatively confirmed the reversibility of the enzyme reaction previously observed with crude enzyme preparations.

Induction of manganese-superoxide dismutase in MRC-5 cells persistently infected with an alphavirus, sindbis.

Sindbis virus (SV), a single-stranded positive-sense RNA virus, multiplies in a variety of cells and causes various outcomes of infection. As we described acute infection of SV induces stress response of small heat shock protein HSP27 and activation of mitogen-activated protein kinase (MAP) signaling pathway (Nakatsue, T., et al., Biochem. Biophys. Res. Commun. 253, 59-64, 1998). In contrast to lytic infection in Vero cells, MRC-5 cells, a human fetus lung cell line, resulted in persistent infection by SV. Here we investigated a cellular factor involved in persistent infection of MRC-5 cells infected with SV. Partial sequence analysis of a 25 kilodalton (kDa) protein, accumulated in large amounts in the cells, showed that manganese-superoxide dismutase (Mn-SOD) was induced during the infections. When Mn-SOD was overexpressed in Vero cells, 20% of the cells survived more than one month, in contrast with the death of 99% of the vehicle-transfected Vero cells at 48 h after infection with SV. These data strongly suggest that a cellular factor which regulates the oxidative pathway modulates the outcome of SV infection.

Surgical management of infants with mitral valve stenosis or atresia without diminutive ascending aorta.

The surgical strategy in infants with mitral valve stenosis or atresia without diminutive ascending aorta remains to be established, including the potential for biventricular repair as a definitive operation. Our surgical experience of six infants with mitral valve stenosis (4 patients) or atresia (2 patients) without diminutive ascending aorta was evaluated based on three important factors: left ventricular volume; the nature of the systemic outflow obstruction; and the type of mitral valve anomaly. Two patients with systemic outflow tract diameter less than 65% of normal underwent systemic outflow tract reconstruction, and the other patients with outflow tract diameter more than 68% of normal were able to maintain systemic circulation without repair. Only one patient with mitral valve stenosis without left ventricular outflow tract obstruction underwent a successful open mitral valvotomy as a biventricular repair after first-stage palliation. The left ventricle of the other patients did not grow after first-stage palliation. Due to progressive subaortic narrowing, pulmonary artery banding should be avoided in patients with mitral atresia due to absent atrioventricular connection who are future Fontan candidates. Most patients with this lesion can be expected to become candidates for safe Fontan-type repair.

Enzymological and molecular biological studies on anandamide amidohydrolase.

Previously we suggested that anandamide amidohydrolase partially purified from porcine brain catalyzed the anandamide synthesis. The reversibility of the anandamide hydrolytic reaction was confirmed with a recombinant enzyme of rat liver. We also showed that the recombinant enzyme had a wide substrate specificity hydrolyzing primary amides and esters of fatty acids in addition to anandamide. When the organ distribution of anandamide amidohydrolase was examined with rats, a large amount of the enzyme was contained in small intestine as well as liver and brain. The intestinal hydrolase was masked by endogenous lipid inhibitors. The enzyme was also found in various eye tissues.

Investigation of the mechanism of lymphocyte injection therapy in treatment of lymphedema with special emphasis on cell adhesion molecule (L-selectin).

We previously employed intraarterial lymphocyte injection therapy in conjunction with standard non-operative treatment of peripheral lymphedema of various etiologies. In this study, we further evaluated the clinical outcome of this therapy in 46 patients with unilateral lymphedema of the extremities. The results showed combined therapy (lymphocyte injection with compression) was effective in 74% (34 of 46 patients) with dramatic reduction in lymphedema in 37% (17 of 46 patients). In the most recent 5 patients treated, we examined the expression of cell adhesion molecule of the lymphocytes (L-selectin) before, during and after lymphocyte injection therapy to study the putative pathomechanism of this treatment method. The expression of L-selectin, a lymphocyte-specific adhesion molecule, increased in the autologous lymphocytes obtained by a blood cell separator and in the lymphocytes from the peripheral blood after injection. Moreover, the lymphocyte fraction, which was positive for L-selectin and negative for CD3, a T-cell marker, decreased after lymphocyte injection. We postulate that the lymphocytes of L-selectin (+) and CD (-) remain in the affected swollen limb and play a role in an ill-defined immunologic responsiveness that potentiates reduction in edema.

Changes in plasma free and sulfoconjugated dopamine in patients with congenital heart disease who underwent cardiac operation.

It has been suggested that plasma sulfoconjugated dopamine (DA) may serve as a source or reservoir for free DA in plasma. Moreover, it has also been reported that the plasma levels of conjugated DA may be used as an index predicting heart failure in patients with heart disease. Therefore, in the present study, we have measured the plasma levels of free and sulfoconjugated DA in patients with congenital heart disease who underwent total corrective operations. The patients were divided into two groups with (6 patients with tetralogy of Fallot, TOF) or without (5 patients with ventricular septal defect without pulmonary hypertension, VSD) cyanosis (mean age of 2.11 years). Blood samples were collected before and after operation from the patients, and plasma free and sulfoconjugated DA levels were measured using high performance liquid chromatography. Preoperative levels of free DA in patients in both groups were higher than the level in age matched control subjects. The plasma level of conjugated DA in TOF was higher than that in the controls and was the highest in VSD before operation. DA infusion early after operation caused a rise in plasma free and conjugated DA, however, the levels of increased free DA were lower in the VSD than in the TOF group. After discontinuing DA infusion, the plasma levels of free DA remained higher, while those of conjugated DA decreased to a level lower than the preoperative values in both groups. As the plasma levels of free and sulfoconjugated DA vary with hemodynamics, it was assumed that the difference in the plasma sulfoconjugated DA level between the groups before operation was due to the influence of pulminary blood flow on catecholamine homeostasis. Since the decrease in conjugated DA has been postulated to be an index of sustained heart failure, it is conceivable that it takes a long time for patients who underwent cardiac operations in infancy to recover from heart failure.

Effect of amiloride on ischaemia and reperfusion injury in isolated, perfused rat hearts.

The effect of amiloride, a potent inhibitor of Na+/H+ exchange, on ischaemic reperfused rat hearts was studied in order to investigate whether Na+/H+ exchange or Na+/Ca2+ exchange is involved in ischaemia-reperfusion injury, When hearts were pre-ischaemically loaded with 100 microM amiloride, recovery of left ventricular developed pressure was significantly better than in control hearts, whereas recovery of heart rate at 30-min reperfusion was unaffected. Amiloride pretreatment also decreased creatine phosphokinase activity in the coronary effluent and completely abolished occurrence of ventricular arrhythmias during reperfusion. It also inhibited intracellular Na+ accumulation early in reperfusion (within 5 min), whereas in the late stage (from 5 to 30 min), Ca2+ overload was inhibited. The findings suggest that Na+/H+ exchange participates mainly in the early stage of reperfusion injury and the Na+/Ca2+ exchange system, secondary to Na+/H+ exchange, in the late stage. The reduction in post-ischaemic cardiac dysfunction induced by amiloride pretreatment may be attributable to inhibition of the resultant Ca2+ accumulation during reperfusion.

Cloning and functional analysis of human p51, which structurally and functionally resembles p53.

The p53 tumor suppressor gene, which is induced by DNA damage and/or stress stimuli, causes cells to undergo G1-arrest or apoptotic death; thus it plays an essential role in human carcinogenesis. We have searched for p53-related genes by using degenerate PCR, and have identified two cDNA fragments similar to but distinct from p53: one previously reported, p73, and the other new. We cloned two major splicing variants of the latter gene and named these p51A and p51B (a human homologue of rat Ket). The p51A gene encodes a 448-amino-acid protein with a molecular weight of 50.9 kDa; and p51B, a 641-amino-acid protein with a molecular weight of 71.9 kDa. In contrast with the ubiquitous expression of p53, expression of p51 mRNA was found in a limited number of tissues, including skeletal muscle, placenta, mammary gland, prostate, trachea, thymus, salivary gland, uterus, heart and lung. In p53-deficient cells, p51A induced growth-suppression and apoptosis, and upregulated p21waf-1 through p53 regulatory elements. Mutations in p51 were found in some human epidermal tumors.

Extracellulary administered lysophosphatidylcholine causes Ca2+ efflux from freshly isolated adult rat cardiomyocytes.

It has previously been reported that ischemia and reperfusion of the heart cause accumulation of lysophosphatidylcholine (LPC) within the myocardium. While it is known that LPC causes the transient increase of intracellular free Ca2+ concentration ([Ca2+]i) during contraction of cardiac cells, little is known about the mechanism for decreasing [Ca2+]i in cardiomyocytes during LPC accumulation. Since cumulative elevation in [Ca2+]i leads to irreversible injury to cardiomyocytes, elevated [Ca2+]i must be restored to an unstimulated level to maintain cell functions. In the present study, we therefore examined the effect of LPC on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. LPC stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner (10(-7)M-10(-5)M). Other lysophospholipids, which are generated from phospholipids of the cell membrane, failed to induce 45Ca2+ efflux from the cells. Dilazep and K-7259, which are known to inhibit the increase in [Ca2+]i caused by LPC, likewise reduced 45Ca2+ efflux caused by LPC addition. Furthermore, the LPC-stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. On the other hand, inhibitors of Na+/Ca2+ exchange, amiloride and 5-(N,N-dimethyl)-amiloride, inhibited LPC induced 45Ca2+ efflux. These results suggest that LPC stimulates extracellular Na(+)-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through Na+/Ca2+ exchange on the plasma membrane of the cells.

Effect of angiotensin II on Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchanger.

In the present study, we examined the effect of angiotensin II on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. Angiotensin II stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner, at least in pharmacological doses of 10(-8) M to 10(-5) M. The 45Ca2+ efflux was inhibited by the type 1 angiotensin II receptor antagonist losartan, but not by the type 2 antagonist PD 123319. Angiotensin II also induced an increase in cytosolic free calcium ([Ca2+]i) and inositol trisphosphate formation within the cardiomyocytes. Angiotensin II-induced 45Ca2+ efflux and the increase in [Ca2+]i were both inhibited by thapsigargin, a specific inhibitor of the sarcoplasmic reticulum Ca2+ pump. The 45Ca2+ efflux was not affected by removal of the extracellular Ca2+ but was dependent on the presence of extracellular Na+. In addition, angiotensin II caused 22Na+ influx into the cells. These results indicate that angiotensin II stimulates Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane type 1 angiotensin II receptors. Angiotensin II-induced increase in [Ca2+]i may cause an activation of Na+/Ca2+ exchange which finally results in the stimulation of 45Ca2+ efflux from the cells. Since it is reported that Na+/Ca2+ exchange is important in calcium homeostasis within the cells, angiotensin II may play some role in the reduction of intracellular Ca2+ from isolated adult rat cardiomyocytes.

Acute infection of Sindbis virus induces phosphorylation and intracellular translocation of small heat shock protein HSP27 and activation of p38 MAP kinase signaling pathway.

In general, viral infection is supposed to induce stress responses in the host cell. However, very few detailed observations about virus-induced stress responses have been reported. Here we investigated specific stress responses in Vero cells infected with Sindbis virus (SV), a single-stranded RNA virus, acute infection with which is known to cause apoptotic cell death in the host cells. Prior to the onset of apoptosis, p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinases (JNKs) were activated. Subsequently, a 27-kDa heat shock protein (HSP27) became phosphorylated, and intracellular distribution of HSP27 was changed from the cytoplasm to the perinuclear region. These results indicate that the cellular signaling cascades activated by pro-inflammatory cytokines and environmental stresses are also activated as a result of lytic infection with SV. These responses may contribute to the delayed onset of apoptosis in the host cells and the facilitation of viral replication.

[A case of reoperation 24 years after repair of absent pulmonary valve syndrome with anomalous origin of the left pulmonary artery].

We experienced a case of a 38-year-old woman with a persistent cough, 24 years after repair of absent pulmonary valve syndrome with anomalous origin of the left pulmonary artery. The right pulmonary artery was massively dilated, thus it caused the compression of the bronchi, which was thought to result in her respiratory symptom. This dilatation of the right pulmonary artery seemed to have progressed because of the following two reasons. The first is the pulmonary hypertension caused by the late reconstruction of the left pulmonary artery. The second is residual pulmonary stenosis and regurgitation after the initial operation without a pulmonary valve insertion. We performed a reoperation consisting of reconstruction of the right ventricular outflow tract using a valved conduit and plication of the right pulmonary artery. Her postoperative course has been without any complications and satisfactory for the past 2 years.

Effect of docarpamine, a novel orally active dopamine prodrug, on the formation of free and sulfoconjugated dopamine in patients who underwent cardiac surgery.

To evaluate the clinical efficacy of orally active dopamine prodrug, docarpamine [N-(N-acetyl-L-methionyl)-O,O-bis (ethoxycarbonyl) dopamine], we examined its effect on the formation of free and sulfoconjugated dopamine in patients who underwent cardiac surgery. The preoperative values of free and sulfoconjugated dopamine in patients were 216 +/- 52 pg/ml and 4,930 +/- 820 pg/ml, respectively. The plasma level of free dopamine increased to 95.3 +/- 28.3 ng/ml by dopamine infusion after the operation and was sustained at a high level (87.7 +/- 26.5 ng/ml) by concomitant administration of docarpamine in spite of tapering of dopamine infusion. After stopping dopamine infusion, plasma level of free dopamine was 24.5 +/- 17.6 ng/ml maintained by oral administration of docarpamine alone. From these results, docarpamine may be a useful alternative to intravenous dopamine after cardiac surgery. Sulfoconjugated dopamine in plasma increased to 267 +/- 120 ng/ml after the start of dopamine infusion and increased further after oral docarpamine administration to 2,060 +/- 610 ng/ml. Since sulfoconjugated dopamine is thought to be a possible precursor of active free dopamine in plasma, orally administered docarpamine might be stored as a reserve pool for free dopamine in patients who undergo cardiac surgery.