Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts.

The cardiac ATP-sensitive potassium (KATP) channel is thought to be a complex composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the sulfonylurea receptor (SUR2). This channel is activated during myocardial ischemia and protects the heart from ischemic injury. We examined the transcriptional expression of these genes in rats with myocardial ischemia. 60 min of myocardial regional ischemia followed by 24-72 h, but not 3-6 h, of reperfusion specifically upregulated Kir6.1 mRNA not only in the ischemic (approximately 2.7-3.1-fold) but also in the nonischemic (approximately 2.0-2.6-fold) region of the left ventricle. 24 h of continuous ischemia without reperfusion also induced an increase in Kir6.1 mRNA in both regions, whereas 15-30 min of ischemia followed by 24 h of reperfusion did not induce such expression. In contrast, mRNAs for Kir6.2 and SUR2 remained unchanged under these ischemic procedures. Western blotting demonstrated similar increases in the Kir6.1 protein level both in the ischemic (2.4-fold) and the nonischemic (2.2-fold) region of rat hearts subjected to 60 min of ischemia followed by 24 h of reperfusion. Thus, prolonged myocardial ischemia rather than reperfusion induces delayed and differential regulation of cardiac KATP channel gene expression.

Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca(2+) overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection.

Mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca(2+) concentration ([Ca(2+)](m)), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoK(ATP) channel opener diazoxide attenuated the accumulation of [Ca(2+)](m) during simulated I/R (EC(50)=18 micromol/L). These effects of diazoxide were blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca(2+)](m) accumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, DeltaPsi(m), in permeabilized myocytes revealed that diazoxide depolarized DeltaPsi(m) (by 12% at 10 micromol/L, P<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca(2+) overload, as a consequence of partial mitochondrial membrane depolarization by mitoK(ATP) channels, underlies cardioprotection. Furthermore, mitoK(ATP) channels and the MPT differentially affect mitochondrial calcium homeostasis: mitoK(ATP) channels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion.

Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells.

Mitochondria can either enhance or suppress cell death. Cytochrome c release from mitochondria and depolarization of the mitochondrial membrane potential (DeltaPsi) are crucial events in triggering apoptosis. In contrast, activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels prevents lethal ischemic injury in vivo, implicating these channels as key players in the process of ischemic preconditioning. We probed the relationship between mitoK(ATP) channels and apoptosis in cultured neonatal rat cardiac ventricular myocytes. Incubation with 200 micromol/L hydrogen peroxide induced TUNEL positivity, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and dissipation of DeltaPsi. Pharmacological opening of mitoK(ATP) channels by diazoxide (100 micromol/L) preserved mitochondrial integrity and suppressed all markers of apoptosis. Diazoxide prevented DeltaPsi depolarization in a concentration-dependent manner (EC(50) approximately 40 micromol/L, with saturation by 100 micromol/L), as shown by both flow cytometry and quantitative image analysis of cells stained with fluorescent DeltaPsi indicators. These cytoprotective effects of diazoxide were reproduced by pinacidil, another mitoK(ATP) agonist, and blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (500 micromol/L). Our findings identify a novel mitochondrial pathway that is protective against apoptosis. The results also pinpoint mitoK(ATP) channels as logical therapeutic targets in diseases of enhanced apoptosis and oxidative stress.

Inhibitory effect of fumaric acid on forestomach and lung carcinogenesis by a 5-nitrofuran naphthyridine derivative in mice.

The inhibitory effect of fumaric acid (FA) on carcinogenesis by potassium 1-methyl-7-[2-(5-nitro-2-furyl)vinyl]-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate (trans, NFN) was examined histologically with male ICR/JCL mice. NFN was fed to mice at a dose level of 0.012% in the diet for 14 weeks. These mice were then divided into 2 groups. One group was given a basal diet, and the other group was given a diet containing 1% FA in the subsequent 39 weeks. In the group of 30 mice fed NFN alone, squamous cell carcinomas were found in the stomachs of 7 mice, multiple papillomas in the stomachs of 13 mice, and multiple and large papillary adenocarcinomas in the lungs of 27 animals. The administration of FA suppressed the NFN-induced stomach and lung carcinogenesis. In the group of 32 mice fed NFN and FA, no stomach tumors developed except 1 early-stage of squamous cell carcinoma. In the lungs, only a small focus of mild atypical hyperplasia and a few early-stage adenocarcinomas were noted in 7 and 11 animals, respectively.

Inhibitory effect of fumaric acid on 3-methyl-4'-(dimethylamino)-azobenzene-induced hepatocarcinogenesis in rats.

Fumaric acid (FA) was examined for its effect on hepatocarcinogenesis in rats fed 3-methyl-4'-(dimethylamino)azobenzene (3-Me-DAB). Male DONRYU rats were given approximately 0.5 g 3-Me-DAB by being fed a diet containing 0.06% 3-Me-DAB for 50 days; they were then given a diet containing 1% FA and drinking water containing 0.025% FA for 51 weeks. The administration of FA effectively suppressed the development of hepatocellular carcinoma, hyperplastic nodules, and hyperplastic areas in the livers of rats fed 3-Me-DAB.

Inhibitory effect of fumaric acid on hepatocarcinogenesis by thioacetamide in rats.

An inhibitory effect of fumaric acid (FA) on hepatocarcinogenesis was examined in rats fed thioacetamide (TAA). A group of male DONRYU rats were fed TAA at a level of 0.035% in the diet for 40 weeks and then fed a basal diet for 40 weeks. Hepatic carcinomas developed in 9 of 41 animals of this group fed TAA alone. The effect of FA on the carcinogenesis was examined in 2 groups fed both TAA and FA; one group of rats were fed FA at 1% in a basal diet after ingestion of TAA, and another group of rats were fed TAA plus a supplement of 1% FA in the diet. The inhibitory effect of FA on TAA carcinogenesis was so marked that no hepatic carcinomas were found in both groups fed FA in combination with TAA.

Analysis of loss of nuclear RNA in azo dye-induced hepatoma by DNA-RNA competitive hybridization.

DNA-RNA hybridization studies, using nuclear RNA's (nRNA's) labeled in vivo and in vitro with high specific radioactivities, were performed to compare the nRNA populations of normal rat liver, livers treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), and 3'-Me-DAB-induced hepatomas. The study with normal liver nRNA labeled by i.p. injection of [3H]orotic acid indicated that the nuclei of a 3'-Me-DAB-induced transplanted hepatoma, AH136B, lacked some RNA species present in normal liver nuclei. No qualitative difference in thee RNA populations was seen between normal liver and the livers of rats fed a carcinogenic amount of 3'-Me-DAB, either alone or in combination with 4-nitrostilbene which enhanced the azo dye carcinogenesis. Then, nRNA's of both normal liver and AH136B hepatoma were labeled in vitro by phosphorylation with polynucleotide kinase and adenosine 5'-[gamma-32P]triphosphate. The competitive hybridization with 32P-labeled normal liver nRNA was competed, and the deletion of RNA in the nuclei of AH136B hepatoma or 3'-Me-DAB-induced primary hepatoma was estimated to be 15% or more in the measure of radioactivity of the hybridized normal liver nRNA. 32P-labeled AH136B hepatoma nRNA was completed completely by liver nRNA's, suggesting that no unique RNA species were present in the hepatoma nuclei.

Inhibitory effect of Capsella bursa-pastoris extract on growth of Ehrlich solid tumor in mice.

The treatment of ICR mice with i.p. injections (0.14 g/kg/day) of the extract of Capsella bursa-pastoris herb (Cruciferae) caused 50 to 80% inhibition of the solid growth of Ehrlich tumor cells that had been inoculated into the s.c. tissue of the animals. The tumor lumps in the treated mice showed multifocal necroses and the infiltartion of host fibrous tissue cells. Experiments were also performed to isolate and identify the active component for the antitumor action, and an acidic substance was isolated in crystalline form from the herb extract. This acidic substance was identified as fumaric acid and was effective in inhibiting the growth of Ehrlich solid tumor at a dose of 10 mg/kg/day. The 50% lethal dose (i.p.) of this acid was 266 mg/kg.

Tissue reactions to calcined bone graft.

Calcined bone blocks made by heating raw bovine bone at 1200 degrees C were implanted into defects in canine femoral condyles. X-ray diffraction patterns showed that the calcined bone was well-crystallized hydroxyapatite. Rapid, good new bone growth was observed around the calcined bone which was gradually absorbed along its surface and also by the Haversian canals.

Induction of hepatic tissue-type plasminogen activator and type 1 plasminogen activator-inhibitor gene expressions and appearance of their translation products in the bile following acute liver injury in rats.

BACKGROUND/AIMS: The plasminogen activator (PA)-plasmin system is primarily involved in fibrinolysis, but is also in the patho/physiological events in which breakdown of extracellular matrices is evoked topically. In this present study, we examined the expression of fibrinolytic factors, tissue-type PA (t-PA) and Type 1 PA inhibitor (PAI-1), in acute liver injury. METHODS: Acute liver injury was produced in rats by the intraperitoneal administration of carbon tetrachloride (CCl(4)). Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured to verify the hepatocellular damage. t-PA and PAI-1 gene expressions were measured by Northern blotting, and the cell type(s) expressing these genes was identified by in situ hybridization. t-PA and PAI-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: A single intraperitoneal administration of CCl(4) caused severe acute parenchymatous liver injury. Both t-PA and PAI-1 gene expressions were induced by the acute liver injury, and plasma t-PA and PAI-1 concentrations were also increased. In situ hybridization studies demonstrated that the hepatocytes were the cells expressing t-PA and PAI-1 genes during the acute liver injury. t-PA was also augmented in the bile, whereas PAI-1 was decreased there. CONCLUSIONS: t-PA and PAI-1 gene expressions are induced in the hepatocytes of rats with acute liver injury. These fibrinolytic factors induced by liver injury may play important roles in liver regeneration.

Effects of aflatoxin B1 on myelopoiesis in vitro.

The short-term cultures of mouse myeloid progenitor cells for granulocytes and monocytes, granulocyte-monocyte colony-forming units (CFU-GM) (CFU-GM assay) and mouse long-term bone marrow culture (LTBMC) were used to investigate the hemopoietic suppression caused by aflatoxin B1 (AFB1). A dose-related suppression of granulopoiesis in short-term bone marrow cultures was seen when the cultures were treated with 10, 5, 1, 0.5 and 0.1 micrograms of AFB1/ml. Two selected doses of AFB1 (5 and 0.5 micrograms/ml) considered to be highly and slightly suppressive in CFU-GM assay exerted a strong suppression of myelopoiesis in LTBMC when applied long-term. Short-term (2 h) exposure of LTBMC to 5 micrograms of AFB1/ml caused a small damage to the myelopoiesis detected in the non-adherent layer. Short-term exposure to 0.5 micrograms AFB1/ml was without any effect on myelopoiesis in LTBMC. The production of colony-stimulating activity (CSA) by an adherent layer of LTBMC was decreased on the second and fifth day after the short-term exposure to both doses of AFB1 and comparable with non-treated culture on the seventh day after the exposure. Presented results indicate that both short-term culture of CFU-GM and LTBMC can be used in the definition and the prediction of host toxicity of AFB1 to hemopoiesis. However, comparing these two in vitro systems, the LTBMC appears to be more sensitive and discriminatory in an evaluation of hemopoietic toxicity.

Cluster analysis on multiple drugs susceptibility supplements genotyping of methicillin-resistant Staphylococcus aureus.

OBJECTIVE: To evaluate the typing power of cluster analysis of antimicrobial susceptibility. METHODS: Results of pulsed-field gel electrophoresis in 71 strains of methicillin-resistant Staphylococcus aureus were compared with cluster analysis of the diameter of growth inhibition in 11 drugs. Subjects were a consecutive series of patients (n = 71) from the wards and outpatient units of a community teaching hospital. RESULTS: The cluster analysis took 2 to 3 seconds once the data were entered into a computer. The sensitivity, specificity, and accuracy of the cluster analysis were 76.3%, 58.3%, and 73.2%, respectively, using genotyping as the reference. CONCLUSIONS: The cluster analysis offered real-time epidemiologic data at minimal cost and labor, warranting its cost-effective role.

Alterations in the growth and cycling status of granulocyte-monocyte colony-forming units (CFU-GM) in rats injected single doses of aflatoxin B1.

To determine the toxic effect of aflatoxin B1 (AFB1) on granulopoiesis in vitro, cultures of myeloid progenitor cells for granulocytes and macrophages CFU-GM in semisolid agar medium were used to evaluate colony formation and tritiated thymidine suicide technique for analysis of cycling status of CFU-GM. Male Fischer rats, 5 to 6 weeks old, were given a single i.p. injection of 1 mg/kg or 0.1 mg/kg of AFB1 which were approximately 1/5 or 1/50, respectively, of LD50 dose of AFB1. The administration of either dose of AFB1 caused a significant reduction of the number of CFU-GM derived colonies on the first and the second day after injection. This reduction was followed by a strong enhancement in CFU-GM colony formation on the third day, and by restoration to the normal level on the fourth day. The increase in percentage of CFU-GM in S-phase preceded one day the peak in CFU-GM colony formation. The two different doses of AFB1 exerted similar effects on the growth and cycling status of CFU-GM. Our data outline an early suppressive effect exerted by AFB1 to the rat granulopoiesis in vivo.

A case of aortic septal defect, associated with patent ductus arteriosus and aberrant right subclavian artery.

This report describes an operation, successfully performed by the authors, on an aorta septal defect (A-P Window) associated with patent ductus arteriosus (PDA) and aberrant right subclavian artery (ARSA). When an A-O Window is complicated by PDA and ARSA, preoperative diagnosis is often difficult. It is often mistaken for a large PDA and ARSA. Therefore at the time of operation of a large PDA and ARSA, it is necessary tao keep artificial heart-lung machine ready so that the A-P Window can be corrected at the same operation if necessary. In this case, the aortopulmonary communication was closed directly by transpulmonary approach under profound hypothermia with extracorporeal circulation after the division of ductus arteriosus and the dissection and mobilization of the stenotic esophagus from surrounding tissues of the ARSA. The division of the ARSA was not carried out, because patient was asymptomatic. The A-P Window associated with PDA and ARSA has apparently not been reported.

Preparation and characterization of double layered coating composed of hydroxyapatite and perovskite by thermal decomposition.

A new modified thermal decomposition method is described for preparing a double layered coating on titanium plates which includes an initial perovskite (CaTiO3) layer followed by a hydroxyapatite (HA) layer on top. The characterization of the coating was studied by X-ray diffractometry and infrared spectroscopy and indicated that the double layer consisted of carbonate HA and CaTiO3 and the thickness of the layer was 4 microns. The coating was performed on the inner surfaces of 50-200 microns sized pores and was also consistent in the smallest of the pores even those of 50 microns. Bone formation was examined in canines at 2-32 week intervals and was dominant on coated plates and in large-sized pores before 16 weeks. However, after 16 weeks bone ingrowth was similar in non-coated and coated plates and in all pore sizes. The results indicated that HA could only influence early bone ingrowth, though good bone ingrowth into small pores indicated that HA exhibited enhanced osteocompatibility. Our methodology ensured the stability of the HA layer consequently minimizing the problems associated with HA loss.

Effect of hydroxyapatite microcrystals on macrophage activity.

Hydroxyapatite (HAp) microcrystals were synthesized by a neutralization reaction of Ca(OH)2 suspension and H3PO4 solution using an ultrasonic homogenizer. The in vitro interaction of HAp microcrystals with rat peritoneal macrophages was investigated by measuring the viability, acid phosphatase (ACP) activity, lactate dehydrogenase (LDH) activity and intracellular calcium content. HAp calcined at 800 degrees C and alpha-alumina particles (alumina) were used as comparative materials. Macrophages actively phagocytosed HAp microcrystals by dissolving them. However, no damage in macrophages exposed to HAp microcrystals was observed by transmission electron microscopy. Macrophages in the presence of HAp microcrystals showed less ACP and LDH activity and higher intracellular calcium content than those in the presence of calcined HAp and alumina. HAp microcrystals had excellent biocompatibility to macrophages as well as sintered HAp.

Effect of exercise on strength and chemical composition of rat femur bone.

The effect of exercise on strength and chemical composition of rat femur bone was examined. Ten 5-week-old rats were forced to exercise on a treadmill with a running speed of 20 m/min at set time intervals for a 4-week period. Another 10 rats were kept idle as controls. After exercising, bending tests of the rat femur bones were carried out, and the content ratios of Sr to Ca, Sr/Ca, were measured by energy-dispersive fluorescent X-ray analysis and EPMA line analysis. The strength of the exercise group was greater than that of the control group. The ratio, Sr/Ca, of the exercise group showed a tendency to increase compared to that of the control group. It is clear that the chemical composition of bone varies with the degree of exercise, and that the bone becomes strengthened at the same time.

Cytotoxicity of synthetic barium hydroxyapatite.

Barium hydroxyapatite (Ba10(PO4)6(OH)2, Ba-HAp) was synthesized by a wet method using Ba(OH)2.8H2O and (NH4)2HPO4 as starting materials. The Ba-HAp obtained had a Ba/P atomic ratio of 1.76 and contained CO3 groups. The Ba-HAp was sintered at 1073 K for 12 hours. The sintered Ba-HAp had a three point bending strength of 29 MPa and Young's modulus of 27 GPa. Cytotoxicity of the sintered bodies and particles was tested using L-cells. The sintered Ba-HAp showed no cytotoxicity, and the cells were closely in contact with the surfaces of sintered Ba-HAp. Morphological observation of the cell around the Ba-HAp particles also showed no cytotoxicity. However, cell growth was inhibited by Ca adsorption on the Ba-HAp particles. These results suggested that the Ba-HAp had no cytotoxicity and can be applied as a bioactive X-ray opaque material.

Effects of Sr-hydroxyapatite microcrystal on cultured cell.

Strontium-hydroxyapatite microcrystals (Sr-HAp sol) were produced by a wet method at room temperature with ultrasonic irradiation and were applied to MC3T3-E1, ROS, and L cells for periods of 2, 4, and 6 days in vitro. The effect on cell growth, the variation of LDH and Ca contents in the media, and attachment between cell and microcrystal were investigated. Sintered Sr-HAp and HAp sol were used as controls. A slight inhibitory effect of Sr-HAp sol on cell growth was found. The degree of inhibition was nearly the same as HAp sol. However, it was stronger than sintered Sr-HAp. The contents of LDH in the media increased with the degree of cell inhibition, and the contents of Ca in the media decreased from the initial stage of cell-sol contact. A good attachment of Sr-HAp sol to cultured cells was seen by phase-contrast microscopy and SEM.

Application of hydroxyapatite-sol as drug carrier.

The application of hydroxyapatite-sol as a drug carrier is being developed. Hydroxyapatite-sol which is a suspension consisting of hydroxyapatite nano-crystals, was synthesized using an ultrasonic homogenizer. The size of the crystals was 40 x 15 x 10 mm3 on average and their specific surface area was 100 m2/g. An amount of a glycoside antibiotics adsorbed onto hydroxyapatite nano-crystals was measured. The drug adsorbed 0.2 mg per 1 mg of hydroxyapatite. The affect of the drug adsorbed onto the hydroxyapatite was investigated using cancer cells. The drug, adsorbed onto the hydroxyapatite nano-crystals, inhibited cancer cell growth.