Effects of Uric Acid on the NO Production of HUVECs and its Restoration by Urate Lowering Agents.

BACKGROUND: Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs). PURPOSE AND METHOD: In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays. RESULTS: HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7 mg/dl for 24 h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect. CONCLUSION: Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.

Carvedilol Suppresses Apoptosis and Ion Channel Remodelling of HL-1 Cardiac Myocytes Expressing E334K cMyBPC.

BACKGROUND: Besides its antiarrhythmic action, carvedilol has an activity to suppress cardiac tissue damage. However, it is unknown whether it has any effect on cellular apoptosis and ion channel remodelling. PURPOSE: To know whether carvedilol has any effect on apoptosis and ion channel remodeling of HL-1 cells expressing E334K MyBPC, and comparing it with bisoprolol. METHOD: We examined effects of carvedilol and bisoprolol on the levels of pro- and anti-apoptotic proteins and ion channels as well as apoptosis of HL-1 cells transfected with E334K MyBPC using Western blot and flow cytometry. RESULTS: Carvedilol decreased the protein levels of p53, Bax and cytochrome c and increased that of Bcl-2 in HL-1 cells expressing E334K MyBPC. Bisoprolol failed to affect the protein levels. Both carvedilol and bisoprolol increased the protein levels of Cav1.2 but not that of Nav1.5. Carvedilol was stronger than bisoprolol at decreasing the number of annexin-V positive cells in HL-1 cells expressing E334K MyBPC. CONCLUSION: Carvedilol suppressed apoptosis of HL-1 cells expressing E334K MyBPC through modification of pro- and anti-apoptotic proteins, whose was associated with an increase of Cav 1.2 protein expression.

Krüppel-like factor 4 mediates anti-proliferative effects of progesterone with G0/G1 arrest in human endometrial epithelial cells.

Activation of the progesterone receptor (PR) inhibits cell proliferation in various reproductive tissues. However, the molecular mechanisms underlying the regulation of cell proliferation by PR remain poorly understood. It is well established that Krüppel-like factor 4 (KLF4), a family of zinc fingercontaining transcription factors, induces cell cycle arrest in epithelial cells. In this study, we investigated whether KLF4 served as a target of PR activation during cell proliferation using human endometrial epithelial cells. PR agonists, progesterone and dienogest, were found to produce a lasting increase in the expression of KLF4 mRNA, followed by a decrease in cyclin D1 mRNA, and inhibit cell proliferation with G0/G1 arrest. KLF4 knockdown using KLF4 small interferingRNA abrogated the inhibition of cell proliferation by PR agonists. In addition, forced expression of KLF4 inhibited cyclin D1 promoter transactivation. These results suggest that PR agonists induce KLF4 expression and then inhibit cyclin D1 expression, and consequently inhibit cell proliferation in human endometrial epithelial cells. In terms of human reproductive tissue, KLF4 may be a factor concerning cell cycle, directly responsive to PR activation.

Functional analysis for peripheral myelin protein PASII/PMP22: is it a member of claudin superfamily?

Two major glycoproteins, P0 and PASII/PMP22, are specifically expressed in peripheral myelin. Point mutations of these proteins and over or under expression of PASII/PMP22 cause various hereditary peripheral neuropathies. P0 is well characterized as a major adhesion molecule in PNS myelin, but the function of PASII/PMP22 is still unknown. Recently, an oligodendrocyte-specific protein (OSP) was identified as a member of the claudin family and as a component of tight junctions of central myelins. Since PASII/PMP22 shows similarity in structure to OSP, which is a tetraspan membrane protein, we speculated if PASII/PMP22 could be a member of claudin superfamily. The primary structure of PASII/PMP22 showed a significant homology of 48% and a 21% identity with the OSP sequence. Exogenous expression of PASII/PMP22 in C6 cells significantly inhibited BrdU incorporation to the cells. The C6 cells stably transfected with PASII/PMP22 cDNA showed no homophilic cell adhesive activity. When dorsal root ganglion (DRG) neurons were cocultured on PASII/PMP22 expressing cells, both neurite extension and branching of DRG neurons were significantly inhibited. These results indicate that PASII/PMP22 may play a role in a turning point of Schwann cell development from proliferation to differentiation. On the other hand, the cells expressing claudin family proteins are reported to show strong cell adhesive activity and an ability to form tight junctions with neighboring cells. For this reason, we currently do not have any functional data supporting that PASII/PMP22 is the member of claudin superfamily.

Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome.

Human chromosome 11p15.5 harbors an intriguing imprinted gene cluster of 1 Mb. This imprinted domain is implicated in a wide variety of malignancies and Beckwith-Wiedemann syndrome (BWS). Recently, several lines of evidence have suggested that the BWS-associated imprinting cluster consists of separate chromosomal domains. We have previously identified LIT1, a paternally expressed antisense RNA within the KvLQT1 locus through a positional screening approach using human monochromosomal hybrids. KvLQT1 encompasses the translocation breakpoint cluster in BWS and patients exhibit frequent loss of maternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster. Here we generated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent with the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal alleles of multiple imprinted loci at the centromeric domain including KvLQT1 and p57(KIP2). The deletion had no effect on imprinting of H19 located at the telomeric end of the cluster. Our findings demonstrate that the LIT1 CpG island can act as a negative regulator in cis for coordinate imprinting at the centromeric domain, thereby suggesting a role for the LIT1 locus in a BWS pathway leading to functional inactivation of p57(KIP2). Thus, the targeting and precise modification of human chromosomal alleles using the DT40 cell shuttle system can be used to define regulatory elements that confer long-range control of gene activity within chromosomal domains.

Efficient modification of a human chromosome by telomere-directed truncation in high homologous recombination-proficient chicken DT40 cells.

Truncation of human chromosomes at desired sites by homologous recombination techniques enables functional and structural analyses of human chromosomes and development of human artificial chromosomes. However, this targeted truncation has been inefficient. We describe here an efficient method for targeted truncation in the chicken DT40 cells with a high homologous recombination rate. The human chromosome 22 was transferred into DT40 cells, where human telomeric repeat (TTAGGG)n was targeted to the LIF locus on the chromosome. Molecular and cytogenetic analyses showed that the predicted truncation at the LIF locus occurred in all of the targeted clones.

Effects of M16209 on insulin secretion in isolated, perfused pancreases of normal and diabetic rats.

We investigated the stimulatory effect of M16209 (1-(3-bromobenzo[b]furan-2-yl-sulfonyl)hydantoin), a novel aldose reductase inhibitor, on insulin secretion using isolated, perfused pancreases of rats. In the pancreases from normal rats, M16209 (100 microM) greatly augmented glucose-stimulated insulin secretion, but showed no effect on unstimulated insulin secretion at 2.8 mM glucose. In contrast, gliclazide (10 microM), a sulfonylurea, strongly enhanced both glucose-stimulated and unstimulated insulin secretion. Sorbinil and epalrestat, potent aldose reductase inhibitors, had no stimulatory effect on insulin secretion. M16209 (100 microM) improved appreciably the decreased insulin response to 22.2 mM glucose and enhanced slightly unstimulated insulin secretion in the pancreases of rats with neonatally streptozotocin-induced, non-insulin-dependent diabetes mellitus (NIDDM). Gliclazide (10 microM), however, failed to affect the pancreases of NIDDM rats. Furthermore, M16209 showed no appreciable effect on ATP-sensitive K(+)-channels in pancreatic beta-cells. These results suggest that M16209, unlike sulfonylureas, selectively enhances glucose-stimulated insulin secretion in both normal and NIDDM rats through a direct action on the pancreas. The site of action remains unknown, but the inhibition of aldose reductase or the ATP-sensitive K+ channels is unlikely to be involved.

Loop and distal actions of a novel diuretic, M17055.

We investigated the mechanism of action of a novel 'high ceiling' diuretic, M17055, in in vivo clearance studies with anesthetized dogs during water diuresis and in vitro microperfusion studies of isolated rabbit renal tubules. In the clearance study, intravenous infusion of M17055 (1 mg/kg per h) decreased free water clearance and increased urinary excretion of Na+ and Cl- to a greater extent than did a maximum dose of furosemide (30 mg/kg per h). With the maximum dose of furosemide, an additional dose of M17055 or hydrochlorothiazide resulted in additional suppression of free water clearance. These results indicate that M17055 has some additional mechanisms of action in the distal nephron. In isolated rabbit cortical thick ascending limb of Henle's loop, M17055 applied to the lumen decreased the lumen positive transepithelial voltage at concentrations over 10(-6) M and suppressed the lumen-to-bath 36Cl- flux at 10(-5) M. In the connecting tubule, M17055 added to the lumen suppressed lumen negative transepithelial voltage in a concentration-dependent manner in a range from 10(-4) to 10(-3) M. The effect of M17055 on transepithelial voltage was also observed in the distal convoluted tubule and cortical collecting duct. Moreover, 10(-3) M of M17055 in the lumen significantly decreased the lumen-to-bath 22Na+ flux in the cortical collecting duct. From these observations, it appears that M17055 acts not only on the thick ascending limb of Henle's loop but also on the distal segments via inhibition of electrogenic Na+ transport.

5-Hydroxydecanoate inhibits ATP-sensitive K+ channel currents in guinea-pig single ventricular myocytes.

We investigated the effect of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the electrical activity of guinea-pig ventricular myocytes. The outward K+ current increased by lowering the intracellular ATP concentration (0.5 mM) was efficiently blocked by 5-hydroxydecanoate when recording in the whole cell configuration with the application of voltage ramps. The increase in the time-independent outward K+ current induced by reducing intracellular ATP to 0 mM was also blocked by 5-hydroxydecanoate (10 or 100 microM) and by tolbutamide (1 mM). Using the single channel recording technique, we found that 5-hydroxydecanoate blocked ATP-sensitive K+ channels when its channel open probability was increased by 1 mM ATP together with 1 mM ADP or by an intracellular pH of 6.6. These conditions are well documented to reflect metabolic changes in the early stages of myocardial ischemic attack. These results suggest that 5-hydroxydecanoate could inhibit ATP-sensitive K+ channels, resulting in an antiarrhythmic effect specifically on ischemic hearts.

Blockade of the ATP-sensitive K+ channel by 5-hydroxydecanoate in guinea pig ventricular myocytes.

Effects of a novel antiarrhythmic agent, 5-hydroxydecanoate (5-HD), were investigated on the electrical activity of the guinea pig ventricular myocytes. The shortening of action potential duration induced by applying iodoacetate (IAA) for 5 to 10 min was reversed completely by 5-HD (100 microM) in the papillary muscle. The single channel current recording in the cell-attached configuration revealed both activation of the ATP-sensitive K+ channel during the treatment with IAA and after depression of the channel by the additional application of 100 microM 5-HD. The quick rundown of the ATP-sensitive K+ channel activity interfered the analysis of the drug effect in the usual inside-out patch configuration. The channel activity in the isolated patch was partially recovered and stabilized by applying a tissue extract, which was prepared from guinea pig ventricle. Under this condition relationship between the 5-HD concentration and the K+ channel open probability was characterized with a K1/2 of 0.16 microM and a Hill coefficient of 0.88. The open- and close-time analysis revealed a decrease of the mean duration of the bursting channel opening and an increase of the interburst time under the effect of 5-HD. The inward-rectifier K+ channel, responsible for the resting K+ conductance, was not affected by 5-HD. It was concluded that the curative effect of 5-HD on the shortened action potential in the IAA-treated myocytes is mediated by the depression of the ATP-sensitive K+ channel.

A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel.

We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.

Paget's disease of the male breast--report of a case and histopathologic study.

We report a 61-year-old male with mammary Paget's disease. Physical examination revealed a slightly exudative erythema at the areola and a reddish, enlarged left nipple. No tumor or left axillary lymph nodes was palpable. He underwent a left modified radical mastectomy. Histologically, there was an intraductal carcinoma in the upper portion of the mammary ducts. The axillary lymph nodes that were examined were free of metastasis. Paget cells had neither estrogen nor progesterone receptors. We speculated that the histogenesis of Paget cells involved carcinoma cells that invaded the epidermis of the nipple.

Mechanisms involved in the effect of M6434 on experimental hemorrhagic shock: I. Effects on myocardial contractility and venous return.

To elucidate the mechanisms of protective effect of M6434 on experimental shock, the authors examined the effects of this compound on the survival time and hemodynamic changes in severely hemorrhagic-shocked dogs. We also examined the effects of M6434 on contractile tension of isolated canine ventricular strips and on venous return in dogs with cardiopulmonary bypass in normal and shock state. Intravenous infusion of M6434 at 10 micrograms/kg/min prolonged survival and maintained mean arterial pressure, cardiac output, and first derivative of left ventricular pressure at higher levels than those in the control group, whereas dopamine (10 micrograms/kg/min) did not significantly affect survival time and hemodynamic parameters. M6434 did not change contractile tension in electrically stimulated canine ventricular strips. M6434 (20 micrograms/kg/min) increased the venous return of dogs with cardiopulmonary bypass in both shock and normal state. Phenylephrine (20 micrograms/kg/min) slightly increased venous return in normal state, but not during shock. Dopamine had no effect at 20 micrograms/kg/min, but it increased venous return in both states at 50 micrograms/kg/min. These results suggest that M6434 may improve the hemodynamic derangement in severe hemorrhagic shock through decreasing venous blood pooling.

Mechanisms involved in the effect of M6434 on experimental hemorrhagic shock: II. Effects on energy metabolism and organ blood flow.

Effects of M6434 on survival time and hepatic energy metabolism of hemorrhagic-shocked rats were examined. Effects of the compound on rat mitochondrial respiration and regional blood flow in hemorrhagic-shocked rats were also studied to clarify the mechanisms of the antishock effects. Intravenous infusion of M6434 (3 or 10 micrograms/kg/min) prolonged the survival time of hemorrhagic-shocked rats. M6434 at 10 micrograms/kg/min significantly suppressed the decline of adenosine triphosphate contents and energy charge of the liver, shifted the blood flow distribution from skin and skeletal muscles to vital organs such as the liver and the heart, and also increased cardiac output in hemorrhagic-shocked rats. The mitochondrial respiration was unaffected by M6434 in vitro (10(-6)-10(-5) M). These data suggest that mechanisms of the beneficial effect of M6434 in hemorrhagic-shocked rats may not be based on the direct activation of energy metabolism, but rather on the redistribution of organ blood flow as well as an increase in cardiac output.

[Echographic diagnosis for deep vein thrombosis of the lower extremities].

Echographic examination of the femoral vein was carried out in two positions: first in supine position and then in standing position, in order to make a diagnosis of deep vein thrombosis (DVT) of the lower extremities. The distance between the wall images of the femoral vein was measured on the line passing through the center of the femoral artery and crossing the common tangent line of the femoral artery and vein at right angle, and the ratio of this distance in supine position to that in standing position was named FEMORAL VEIN DISTENSIBILITY INDEX (FVDI). The FVDI was 2.83 +/- 0.83 in control group consisting of 14 healthy subjects, and 1.26 +/- 0.18 in DVT group consisting of 21 patients with 23 legs. There was a statistically significant difference between the two groups (p less than 0.001). No statistical difference of the FVDI was, however, noted between the control group and the diseased group consisting of 19 contralateral, symptom-free legs. And no relation was confirmed between FVDI and venographical findings, namely site and extension of thrombotic occlusion. By introducing the FVDI, which reflects functional hemodynamics of the femoral vein, the echographic diagnosis for DVT has become easier and more accurate, presenting a diagnostic accuracy of 90.6% in this study.

[Study of mechanism and effect of sodium 5-hydroxydecanoate on experimental ischemic ventricular arrhythmia].

We investigated the effects of sodium 5-hydroxydecanoate (5-HD) on experimentally induced ischemic arrhythmia and its mechanisms of action by biochemical and electrophysiological techniques. 5-HD, at the single dose of 200 mg/kg (p.o.) or at the one week multiple doses of 3 to 100 mg/kg (p.o.), suppressed the incidence of ventricular fibrillation induced by coronary ligation in rats. 5-HD at the dose of 3 or 10 mg/kg (i.v.) elevated the ischemically decreased ventricular fibrillation threshold in the coronary ligated dogs. In isolated rat heart, 5-HD suppressed the K+ release from ischemic myocardium at the doses of 10(-5) to 10(-3) M. 5-HD at the dose of 10(-4) M decreased the open state probability of ATP regulated K+ channel in isolated myocardial cell of guinea pig. Contents of high-energy-phosphate compounds were markedly decreased in ischemic myocardium of rats, and they were not affected by 5-HD. These results demonstrate the efficacy of 5-HD against experimental ischemic ventricular arrhythmia. Its antiarrhythmic action may be attributed, at least in part, to the suppression of K+ release from ischemic myocardium by possibly inhibiting the ATP regulated K+ channel.

[Liposarcomatous tumor emboli to the left iliac artery growing intraluminally into the femoral artery. A report of an operative and autopsy case].

A 74-year-old female patient with a past history of the locally recurrent tumor of the liposarcoma in the right thigh over 20 years ago received an emergency vascular operation to relieve severe ischemic symptoms in the left leg. The left femoral artery was pulseless and was filled with several long, translucent and cord-like tumors which resembled the hands of a cuttlefish. The tumors were easily removed except its root adhering to the left common iliac artery. A crossover femoro-femoral artery bypass was carried out. On the 24th postoperative day she died suddenly. The autopsy findings were as follows: A hen egg-sized tumor occupied almost whole space of the left atrium, and connected with the metastatic right lung tumor via the pulmonary vein. The sudden death was probably due to the abrupt obstruction of the mitral valve by the tumor in the left atrium. The residual tumor was noted in the left common iliac artery, but its adventitia was not involved. The pathohistological diagnosis of each tumor was same, well differentiated liposarcoma. The tumors which occluded the left iliofemoral artery must have originated from the tumor in the left atrium in form of the tumor embolism, lodged in the left common iliac artery, grown intraluminally into external iliac and femoral artery, and finally caused ischemia of the left leg.

[A case of abdominal aortic aneurysm ruptured into the left common iliac vein with symptoms resembling deep vein thrombosis].

A 59 years old male with abdominal aortic aneurysm ruptured into the left common iliac vein was transferred to us with symptoms resembling deep vein thrombosis of the left lower extremity, such as leg pain, swelling and dilatation of the superficial veins. At operation, a Fogarty's occlusion catheter was inserted through the right greater saphenous vein into the inferior vena cava and inflated concomitantly during aortic clamp to prevent pulmonary embolism which may be caused by the dislodged thrombi from the aneurysm, and as well as to control back-flow-bleeding from the central. The fistula (3.0 X 1.0cm) was closed from inside of the aneurysm using the inferior wall of the aneurysm. The blood from the fistula was collected by the Cell Saver and re-transfused to the patient. The abdominal aorta was replaced with a Dacron Y-shaped prosthesis. The postoperative course was uneventful. CTR on chest X-ray subsided from 51% to 42%, cardiac output normalized from 11l/min to 6l/min, and symptoms resembling the deep vein thrombosis disappeared.

Spontaneous remission of hypercalcemia in a functioning parathyroid cyst.

We treated a patient with a hyperfunctioning parathyroid cyst detected incidentally and which disappeared spontaneously. High levels of plasma PTH disappeared after removal of the cystic lesion of the parathyroid gland. Histologically, a secondary pseudocyst resulting from a cystic degeneration of an adenoma was observed. Minor hemorrhage of an unknown cause, within the adenoma, led to the large cystic lesion of the parathyroid gland.

[Pathological and clinical studies on primary aldosteronism].

In the surgical treatment of patients with primary aldosteronism, differentiation of idiopathic hyperaldosteronism due to bilateral adrenal hyperplasia should be kept in mind. Controversies exist, on the other hand, concerning the diagnosis and management of multiple nodular hyperplasia which often coxists with adrenal adenoma. We studied correlations between pathological findings and clinical diagnosis on 13 patients with primary aldosteronism who were operated on during the 19 year period from 1963 to 1981, and the results were as follows; (1) Histologically, adrenal glands of these patients were classified into three subgroups, namely i) adrenocortical adenoma alone, ii) adrenocortical adenoma associated with multiple nodular hyperplasia, and iii) nodules of multiple nodular hyperplasia alone. (2) Clinically, the latter two subgroups showed increase in plasma renin activity following the combination test of volume depletion and spironolactone administration. From these results it is suggested that even in patients with primary aldosteronism multiple nodular hyperplasia of the adrenal cortex can coexist together with adrenal adenoma, and that coexistence of this hyperplasia may modify some clinical features of the disease.