Transfection of NF-κB decoy oligodeoxynucleotide suppresses pulmonary metastasis by murine osteosarcoma.

Nuclear factor-kappa B (NF-κB) has a pivotal role in the progression and distant metastasis of cancers, including malignant bone tumors. To inhibit NF-κB activation, a new molecular therapy using synthetic double-stranded oligodeoxynucleotide (ODN) as a 'decoy' cis element against NF-κB has been developed. To determine whether pulmonary metastasis of osteosarcoma is reduced by inhibiting the action of NF-κB, NF-κB decoy ODN was transfected into the nuclei of murine osteosarcoma cells with high pulmonary metastatic potential, the LM8 cell line, using a three-dimensional alginate spheroid culture model. An in vitro study demonstrated the successful transfection of LM8 cells cultured in alginate beads by 'naked' NF-κB decoy ODN and that the activation of NF-κB signaling was significantly suppressed. Tumor growth was not affected by transfection of NF-κB decoy ODN, however, the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) mRNA was markedly decreased. Furthermore, the transfection of 'naked' NF-κB decoy ODN effectively suppressed pulmonary metastasis in an in vivo alginate bead transplantation model. Our results suggest that NF-κB has a central and specific role in the regulation of tumor metastasis and could be a molecular target for development of anti-metastatic treatments for osteosarcoma.

Inhibitory effect of MS-153 on elevated brain glutamate level induced by rat middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: In this study we investigated the effects of a novel compound, MS-153 ([R]-[-]-5-methyl-1-nicotinoyl-2-pyrazoline), on elevated brain glutamate concentrations and cerebral infarct volume induced by middle cerebral artery (MCA) occlusion in the rat. METHODS: The rat MCA was occluded by a thrombus induced by a photochemical reaction between green light and the photosensitizer dye rose bengal, which causes endothelial injury followed by formation of a platelet- and fibrin-rich thrombus at the site of photochemical reaction; this method is routinely used in our laboratory to produce arterial occlusion in experimental animals. Extracellular glutamate concentration at the ischemic border zone was determined by a microdialysis technique. The size of cerebral infarction was measured by a histochemical technique 24 hours after MCA occlusion. MS-153 was administered at various doses as a continuous infusion for 24 hours, beginning 0 to 2 hours after MCA occlusion. RESULTS: At the ischemic border zone, the concentration of glutamate in the extracellular fluid increased by 40-fold after ischemia. At 3.13 mg/kg per hour, MS-153 reduced glutamate concentration (P < .05) and also the size of ischemic cerebral infarction (P < .05). Furthermore, the glutamate uptake inhibitor DL-threo-beta-hydroxyaspartate reversed the effect of MS-153 on glutamate concentration. CONCLUSIONS: The reduction in the size of cerebral infarction by MS-153 may be attributable to the inhibition of glutamate release or an increase in cellular glutamate uptake.

Glutamate efflux via the reversal of the sodium-dependent glutamate transporter caused by glycolytic inhibition in rat cultured astrocytes.

[3H]L-Glutamate uptake in cultured rat astrocytes was completely reduced by 30 min preincubation with 1 mM of iodoacetic acid, a glycolytic inhibitor. This treatment significantly reduced the energy charge potential, but did not cause membrane destruction in the cultured astrocytes. To examine the effect of iodoacetic acid on the glutamate release, [3H]L-glutamate was preloaded into astrocytes in the presence of methionine sulfoximine, a glutamine synthetase inhibitor, and the total intracellular radioactivity was measured after 30-min treatment with 1 mM iodoacetic acid for comparison with non-treated astrocytes. During the treatment, about 40% of the total intracellular glutamate content was effluxed. This efflux could be decreased by reducing the extracellular potassium ion concentration. The intracellular sodium concentration, measured with a sodium ion-sensitive fluorescent probe (sodium-binding benzofuran isophtalate), gradually increased to 30 mM on addition of 1 mM iodoacetic acid. These results indicate that the glutamate efflux via reversal of the Na(+)-dependent transporter occurred during glycolytic inhibition, and which may be caused by intracellular Na+ overload. Such an iodoacetic acid-induced Na+ overload could be completely diminished by pretreatment with 1 microM 5-[N- ethyl-N-isopropyl]amiloride, a selective Na(+)-H+ antiporter inhibitor, but even this did not stop the iodoacetic acid-induced glutamate efflux. The intracellular pH, measured by a pH-sensitive fluorescent probe [2',7'-bis(carboxyethy)-5,6-carboxy-fluorescein], was gradually decreased to 7.1 by the iodoacetic acid treatment. On the other hand, iodoacetic acid-induced intracellular acidosis was more rapid and severe in the presence of 5-[N-ethyl-N-isopropyl]amiloride. These results suggest that the reversal of the Na+-dependent glutamate transporter may be caused by not only intracellular Na+ overload but also intracellular acidosis.

Pharmacological studies on a new dihydrothienopyridine calcium antagonist. 4th communication: prophylactic and therapeutic effects of S-(+)-methyl-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2, 3- b]pyridine-5-carboxylate in stroke-prone spontaneously hypertensive rats.

The prophylactic and therapeutic effects of S-312-d (S-(+)-methyl-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2, 3- b]pyridine-5-carboxylate, CAS 120056-57-7) were compared with those of nimodipine or nicardipine using male stroke-prone spontaneously hypertensive rats (SHRSP). The survival rate of SHRSP was dose-dependently increased by once a day oral administration of S-312-d (0.3, 1, and 3 mg/kg) or nimodipine (10 mg/kg), while all non-treated SHRSP fed with high Na+ diet died within 40 days after the start of the experiment. All SHRSP treated with 3 mg/kg S-312-d survived during the 60-day experiment periods. Marked decreases of body weights and various neurological symptoms were also inhibited with S-312-d or nimodipine. Moderate diuretic effects were observed with S-312-d at doses of 1 and 3 mg/kg. The appearance of urinary occult blood in control SHRSP was markedly inhibited with S-312-d at 1 mg/kg and nimodipine at 10 mg/kg. Histological examination of the brain of SHRSP showed that cerebral stroke lesion including edema, hemorrhage, and/or softening was dose-dependently inhibited with S-312-d. Once a day oral administration of S-312-d (1, 3, or 10 mg/kg) dose-dependently increased the body weights and improved the neurological symptoms of diseased SHRSP. The appearance of proteinuria and of occult blood in the urine of SHRSP were also markedly inhibited with S-312-d or nicardipine. Histological examination of the brain of SHRSP showed that the arbitrary neurotoxic index (ANI) for stroke lesion dose-dependently decreased with S-312-d at 1, 3, and 10 mg/kg as follows: 4.8, 3.0, 2.3. The ANI for non-treated SHRSP was 7.6. The therapeutic effects of nicardipine (ANI 3.9) at 10 mg/kg corresponded to those of S-312-d at 3 mg/kg. Thus, S-312-d can be recommended for the treatment of cerebral insufficiency or vasospasm following stroke as well as in essential hypertension.

Effects of a novel calcium antagonist, S-(+)-methyl-4,7-dihydro-3-isobutyl-6- methyl-4-(3-nitrophenyl)thieno[2,3-b]pyridine-5-carboxylate (S-312-d), on ischemic amino acid release and neuronal injury in stroke-prone spontaneously hypertensive rats.

The effects of a novel dihydrothienopyridine Ca antagonist S-(+)-methyl-4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitrophenyl)-thieno [2,3-b]pyridine-5-carboxylate (S-312-d), on the amount of amino acid release during cerebral ischemia and delayed neuronal death in the hippocampal CA1 region of stroke-prone spontaneously hypertensive rats were studied and compared with those of nimodipine. The released amino acids were measured by high-performance liquid chromatography after microdialysis. Cerebral ischemia was produced by occlusion of the bilateral common carotid arteries for 20 min. Intraduodenal administration of 0.3 mg/kg of S-312-d at 60 min before the carotid occlusion significantly decreased the ischemic release of glutamate and taurine, but did not influence their basal release. However, nimodipine did not inhibit the ischemic glutamate release even at a dose of 10 mg/kg. Similar peripheral hemodynamic effects were observed before and during bilateral carotid occlusion in groups treated with S-312-d or nimodipine. During the carotid occlusion, almost no cerebral blood flow was observed in either group. Therefore, the inhibitory effect of S-312-d on ischemic amino acid release probably arises from its potent direct action on neuronal cells. The neuronal cell densities of the CA1 subfield at 7 days after 20-min bilateral carotid occlusion significantly decreased in the vehicle-control group compared with the sham-operated group. Intraperitoneal administration of 0.1 mg/kg of S-312-d at 60 min before ischemia prevented the decrease of neuronal cell density compared with the vehicle control. These results show that S-312-d can offer marked neuronal protective effects against ischemic injury.

Changes in extracellular concentration of amino acids in the hippocampus during cerebral ischemia in stroke-prone SHR, stroke-resistant SHR and normotensive rats.

To investigate the role of glutamate release in cerebral ischemia, the amounts of amino acids (glutamate, taurine, alanine, glycine and glutamine) released in the hippocampal CA1 region of stroke-prone SHR (SHRSP), stroke-resistant SHR (SHRSR) and normotensive rats (WKY) were determined during and after cerebral ischemia by the microdialysis method under halothane anesthesia. Cerebral ischemia was produced by the occlusion of both common carotid arteries for 20 min. The basal amino acids release did not differ among the three strains of rats, but ischemic glutamate and taurine releases were more marked in SHRSP than in other strains. These results suggest that the massive glutamate release during cerebral ischemia of SHRSP might be related with severe neuronal cell injury.

Decrease of Na(+)-Ca2+ exchange activity by ascorbate in rat brain membrane vesicles.

Na(+)-dependent Ca2+ uptake in rat brain microsomal membrane vesicles was inhibited by preincubating the vesicles with ascorbic acid at 0.1-10 mM. The inhibitory effect of ascorbate was blocked by simultaneous addition of ascorbate oxidase. The decrease in activity was not reversed upon removing the ascorbate. The kinetic study showed that the treatment with ascorbate decreased Bmax without a change in Km for Ca2+. The inhibitory effect by ascorbate was also observed in membrane vesicles derived from osmotically shocked synaptosomes and in reconstituted membrane vesicles. The effect by ascorbate was specific: it did not affect either ATP-dependent Ca2+ uptake in the presence of o-phenanthroline, an inhibitor of lipid peroxidation, or Na(+)-dependent glutamate uptake in the membrane vesicles. The activity of Na(+)-Ca2+ exchange was also decreased by isoascorbic acid, but not by ascorbate 2-sulfate at 1 mM. The treatment with glutathione or 2-mercaptoethanol did not affect the Na(+)-Ca2+ exchange activity, while 1 mM dithiothreitol caused the inhibition which was completely blocked by o-phenanthroline. The effect of ascorbate on Na(+)-dependent Ca2+ uptake was observed even under the conditions which suppress peroxidation of membrane phospholipids.

Determination of 2,5-hexanedione and N-methylformamide in urine by gas chromatography.

Methods for the clinical testing of 2,5-hexanedione and N-methylformamide, urinary metabolites of n-hexane and N,N-dimethylformamide, respectively, were established by capillary column GLC. Recovery rates of 2,5-hexanedione and N-methylformamide from urine were 80.2-108.9% and 99.6-104.2%, respectively. Coefficients of variation (C.V.) for the amount of each product added were 3.1%, 1.8% (intra-assay) and 5.7%, 3.3% (inter-assay), respectively. The concentration ranges of metabolites from urine of healthy subjects without exposure to these organic solvents were less than or equal to 0.5 mg/l (2,5-hexanedione), less than or equal to 1.5 mg/l (N-methylformamide), respectively.

Inhibition by taurine of Na(+)-Ca2+ exchange in sarcolemmal membrane vesicles from bovine and guinea pig hearts.

1. Taurine, but not GABA, beta-alanine and glycine, inhibited Na(+)-dependent Ca2+ uptake in bovine cardiac sarcolemmal membrane vesicles in a dose-dependent manner. 2. The inhibition of Na(+)-dependent Ca2+ uptake was noncompetitive with respect to Ca2+ concentration. 3. The inhibitory effect of taurine on the exchange was also observed in cardiac sarcolemmal vesicles prepared from guinea pig, but not from rat. 4. Taurine did not affect Na(+)-dependent Ca2+ efflux nor ATP-dependent Ca2+ uptake in the bovine cardiac membranes.

A new micromethod for colorimetric determination of zinc in serum.

A simple micromethod with a colorimetric reagent of high sensitivity was developed for the determination of zinc in serum. For the color reagent 2-(2-pyridylazo)-5-dimethylaminophenol (PADAP) was used. Coefficients of variation of the within-day precision were 6.65 and 2.24% for zinc standard at concentrations of 0.1 and 0.5 microgram/ml, respectively. The mean recovery by this method was 101.2% (S.D.: 3.56%). Zinc concentrations measured by the above method showed good agreement with those by atomic absorption spectrophotometry, with a correlation coefficient of 0.934. Serum zinc values of 50 healthy adults (38 females, 12 males) ranged from 63 to 116 microgram/100 ml (mean: 89.68 microgram/100 ml, Sd.: 13.28 microgram/100 ml).