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Heated scallop-shell (HSS) nano-particles, prepared using a wet grinding mill, and microparticles were examined for their antibacterial activity against vegetative bacterial cells and spores. The median diameters of the nano-particles and micro-particles were approximately 20 nm and 30 µm, respectively. The antibacterial activity of HSS against Escherichia coli increased with an increase in concentration, regardless of particle size; however, the antibacterial activity of the nano-particles was much higher than that of micro-particles. The sporicidal activity of the nano-particles was also much higher than that of micro-particles, with HSS nano-particles able to kill Bacillus subtilis spores. A reduction of more than three orders of magnitude for B. subtilis spores was confirmed following a 30 min treatment at 5 mg/ml and 60℃, showing that the combination of HSS nano-particle treatment with mild heating was particularly effective for controlling bacterial spores.

Selective peroxisome proliferator-activated receptor gamma (PPARγ) modulators are expected to be a novel class of drugs improving plasma glucose levels without PPARγ-related adverse effects. As a continuation of our studies for (-)-Cercosporamide derivatives as selective PPARγ modulators, we synthesized substituted naphthalene type compounds and identified the most potent compound 15 (EC(50) = 0.94 nM, E(max) = 38%). Compound 15 selectively activated PPARγ transcription and did not activate PPARα and PPARδ. The potassium salt of compound 15 showed a high solubility and a good oral bioavailability (58%). Oral administration of the potassium salt remarkably improved the plasma glucose levels of female Zucker diabetic fatty rats at 1 mg/kg. Moreover, it did not cause a plasma volume increase or a cardiac enlargement in Wistar-Imamichi rats, even at 100 mg/kg.

This study was conducted to investigate whether or not a food substitute (Dr. BAANs(R)) containing three bioactive components L-arginine, omega-3 polyunsaturated fatty acid, and ribonucleic acid, supplied orally to 15 overweight patients, may have efficacy to prevent or improve the metabolic syndrome of these patients. To provide supporting data for this clinical study, the in vivo fatty acid metabolism of obese mice was analyzed using (125)I labeled 15-(p-iodophenyl)-9-methylpentadecanoic acid (9MPA) in the tissues' lipid pool. After 3 months of intervention, the results showed that there were improvements observed in liver functions, lipid profiles and metabolic syndrome marker. Significant differences were also found in the values of blood pressure, body weight, percentage of body fat, and body mass index. In the animal study, the tissue uptake of (125)I-9MPA at 10 min after injection was higher in obese mice than in the control mice and the treatment with Dr. BAANs(R) in obese mice decreased the uptake significantly. The final product metabolite of p-iodophenylacetic acid in obese mice was increased significantly by the treatment. In conclusion, this food substitute may have a beneficial effect for the prevention or improvement of metabolic syndrome.

Aptamers are an attractive candidate for a molecular-targeted drug since they can show ability to precisely recognize proteins with a large affinity similar to antibodies. A drawback of aptamers is...

Intrathecal (i.t.) administration into mice of S -(+)-fenfluramine (0.01–0.1 nmol), a serotonin (5-hydroxytryptamine, 5-HT) releaser, produced a behavioral response consisting of scratching, biting and licking. Here, we report the behavioral characteristics and the involvement of interactions between 5-HT and substance P (SP) systems in the S -(+)-fenfluramine-induced behavioral response. The S -(+)-fenfluramine-induced behavioral response peaked at 5–15 min and almost disappeared at 20 min after injection. The behavior induced by S -(+)-fenfluramine (0.1 nmol) was dose-dependently inhibited by an intraperitoneal injection of morphine (0.02–0.5 mg/kg), suggesting that the behavioral response is related to nociception. The S -(+)-fenfluramine-induced nociceptive behavior was significantly inhibited by pretreatment with 5-HT antiserum and co-administration of ketanserin, a selective 5-HT2 receptor antagonist. However, WAY-100635, a selective 5-HT1A receptor antagonist, and ramosetron, a selective 5-HT3 receptor antagonist, were not active. On the other hand, SP antiserum and RP67580, a selective neurokinin-1 (NK1) receptor antagonist, significantly inhibited S -(+)-fenfluramine-induced nociceptive behavior. These results suggest that i.t.-administered S -(+)-fenfluramine releases SP through the activation of 5-HT2 receptors subsequent to 5-HT release, and, as a result, produces nociceptive behavior.

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-d-aspartate receptor mechanism. Brain Res 2002;952:7-14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

The inhibitory effect of repetitiously administered loperamide, a peripheral mu-opioid receptor agonist and well-recognized antidiarrheal agent, on mouse gastrointestinal transit was compared with that of morphine in order to examine the development of tolerance to mu-opioid receptor agonist-induced constipation (antitransit effect). When administered subcutaneously 15 min before the oral injection of charcoal meal, loperamide (0.1-30 mg/kg) and morphine (1-8 mg/kg) dose-dependently and significantly inhibited gastrointestinal transit of charcoal with the ID(50) values of 1.6 (0.3-7.1) mg/kg and 3.6 (1.5-8.5) mg/kg, respectively. When loperamide (30 mg/kg) was administered twice daily for 2 days, the antitransit effect was significantly reduced. On the other hand, morphine did not develop tolerance in even more severe conditions than those of loperamide. It is known that P-glycoprotein, an ATP-dependent drug efflux pump, is involved in the development of tolerance to morphine analgesia. The tolerance observed with loperamide was significantly prevented by cyclosporin (30 mg/kg, i.p.), a P-glycoprotein inhibitor, thus the ID(50) value in loperamide-tolerant mice was markedly reduced from >1000 mg/kg to 40 (2.7-603.0) mg/kg by cyclosporin. These results indicate that loperamide, different from morphine, readily develops tolerance to the inhibitory effect on mouse gastrointestinal transit, and the P-glycoprotein may be involved in the development of tolerance to the antitransit effect of loperamide.

A series of our studies have shown that formation of cholesterol-supersaturated bile in patients with cholesterol gallstone disease is causatively related to decreased gallbladder contractility and mucin hypersecretion by the gallbladder. Supersaturated bile may modify the composition of gallbladder membranes so that the transduction of smooth muscle regulatory signals is impaired, and it may enhance the inflammation-induced mucin secretion by the gallbladder. To achieve a better understanding of the mechanism by which supersaturated bile impairs the contractility, we studied changes in the expression levels of gallbladder cholecystokinin (CCK-A) receptor messenger ribonucleic acid (mRNA) in prairie dogs fed a high-cholesterol diet. Levels of pathobiological determinants in arachidonate metabolism which are important for mucin secretion were also measured in their bile. Adult male prairie dogs were randomly assigned to receive either a semisynthetic diet (SSD) or an SSD plus 1.2% cholesterol (a high-cholesterol diet) for 2-, 4-, and 6-week periods. The contractile force in response to CCK-octapeptide (CCK-8) was measured by using gallbladder muscle strips. The mRNA levels of the CCK-A receptor were determined by reverse-transcription polymerase chain reaction (RT-PCR). Parallel to the increase in the cholesterol saturation index, the contractile responses to CCK-8 decreased in the animals fed a high-cholesterol diet for 4 weeks and markedly decreased in the animals with gallstone formation. However, in contrast to the decreased contractility, the steady-state mRNA levels of the gallbladder CCK-A receptor were significantly increased in the animals fed a high-cholesterol diet in comparison with the corresponding control animals. In the bile, a high-cholesterol diet caused an increase in the proportion of arachidonyl-phosphatidylcholine species, where phospholipase A(2) activity, prostaglandin E(2), and mucin concentrations were increased parallel to the feeding period. Up-regulation of the CCK-A receptor mRNA in the gallbladder of animals fed a high-cholesterol diet associated with decreased contractility may be due to an impairment of CCK signaling related to increased membrane cholesterol contents and its related reaction of biological compensation in order to increase the receptor concentration. The results of the present study suggest that in prairie dogs fed a high-cholesterol diet both a decrease in gallbladder contractility related to impairment of CCK signaling and phospholipase A(2) (PLA(2))-induced mucosal inflammation in the gallbladder with associated biliary alterations favoring cholesterol crystal formation pathogenetically contribute to the formation of cholesterol gallstones.

大腸脂肪腫は比較的稀な粘膜下腫瘍であるが径が大きくなるにつれ,腸重積を合併して発見される頻度が高い.今回われわれは術前にCT,内視鏡検査にて脂肪腫の診断を得て腹腔鏡補助下手術を選択できた症例を経験したので報告する.症例は77歳,男性.左下腹部痛にて近医を受診,注腸造影にて下行結腸下端に表面平滑な円形腫瘤とcoiled spring signを認め,腸重積の診断にて当院紹介となる. CTにて下行結腸内腔に径約3cmの内部が-93HDと脂肪組織に近い濃度のCT値を示す円形の腫瘤を認めた.内視鏡検査ではcushionsignを示す柔らかな粘膜下腫瘍の所見を示しており大腸脂肪腫と診断,腹腔鏡補助下に結腸部分切除を行った.病理組織検査では5×2.5×2.7cm大の成熟した脂肪腫で,漿膜下より発生し腸管内腔に向かって亜有茎性,気球状の発育を示していた.巨大な大腸脂肪腫で術前に質的診断が得られた症例は侵襲の少ない腹腔鏡補助下手術の良い適応であると考えられる.

We have developed a new micro-discharge plasma (MDP)-based gene transfection method, which transfers genes into cells with high efficiency and low cytotoxicity; however, the mechanism underlying the method is still unknown. Studies revealed that the N-acetylcysteine-mediated inhibition of reactive oxygen species (ROS) activity completely abolished gene transfer. In this study, we used laser-produced plasma to demonstrate that gene transfer does not occur in the absence of electrical factors. Our results show that both electrical and chemical factors are necessary for gene transfer inside cells by microplasma irradiation. This indicates that plasma-mediated gene transfection utilizes the synergy between electrical and chemical factors. The electric field threshold required for transfection was approximately 1 kV m−1 in our MDP system. This indicates that MDP irradiation supplies sufficient concentrations of ROS, and the stimulation intensity of the electric field determines the transfection efficiency in our system. Gene transfer by plasma irradiation depends mainly on endocytosis, which accounts for at least 80% of the transfer, and clathrin-mediated endocytosis is a dominant endocytosis. In plasma-mediated gene transfection, alterations in electrical and chemical factors can independently regulate plasmid DNA adhesion and triggering of endocytosis, respectively. This implies that plasma characteristics can be adjusted according to target cell requirements, and the transfection process can be optimized with minimum damage to cells and maximum efficiency. This may explain how MDP simultaneously achieves high transfection efficiency with minimal cell damage.

Acquired drug resistance is a major factor in the failure of doxorubicin-based cancer chemotherapy. We determined the ability of cinchonine to reverse doxorubicin drug resistance in a doxorubicin-resistant leukaemia cell line (mouse P388/DOX). A non-cytotoxic concentration of cinchonine (10 μM) increased the sensitivity to doxorubicin of multidrug-resistant P388/DOX cells and significantly enhanced the doxorubicin-induced apoptosis and DNA fragmentation in resistant cells, but had no effect in parent cells. Time-course studies demonstrated that DNA fragmentation was present 24 h after incubation with doxorubicin and cinchonine, indicating that DNA degradation was a preceding event. In cultured cells, cinchonine increased the intracellular accumulation of doxorubicin in the resistant cells in a dose-dependent manner. Using flow cytometry to measure the inhibition of the P-glycoprotein (P-gp) dependent efflux of rhodamine 123, cinchonine was found to be considerably more effective than quinine. The results with cinchonine suggest that there may be quinine derivatives with a similar capacity to inhibit drug transport by P-gp. Additionally, the G2/M phase cell population in resistant cells is increased by doxorubicin/cinchonine treatment. Exposure of resistant cells to 1 μM doxorubicin and 10 μM cinchonine resulted in the expression of Fas (APO-1/CD95) in cells after 6 h. These studies demonstrate that the cell killing effects of doxorubicin and cinchonine in resistant cells are mediated, at least in part, by the induction of apoptosis.

Anesthetized open-chest dogs were subjected to 15-min myocardial ischemia followed by 2-h reperfusion to induce myocardial stunning. A novel Na(+)/H(+) exchange inhibitor 6,7,8,9-tetrahydro-2-methyl-5H-cyclohepta[b]pyridine-3-carbonylguanidine maleate (TY-12533), administered 10 min before or 10 min after start of ischemia (3 mg/kg/10 min, i.v.), did not affect reductions in regional myocardial wall thickening, blood flow and pH during ischemia, but it significantly improved recovery of the wall thickening and blood flow after reperfusion. These results indicate that TY-12533, even when administered during ischemia, could prevent myocardial stunning without affecting myocardial dysfunction or acidosis induced by brief ischemia.

We elucidated the interaction of small-conductance Ca2+-activated K+(SKCa) channels and L-type Ca2+channels in muscarinic receptor-mediated control of catecholamine secretion in the isolated perfused rat adrenal gland. The muscarinic agonist methacholine (10–300 μM) produced concentration-dependent increases in adrenal output of epinephrine and norepinephrine. The SKCachannel blocker apamin (1 μM) enhanced the methacholine-induced catecholamine responses. The facilitatory effect of apamin on the methacholine-induced catecholamine responses was not observed during treatment with the L-type Ca2+channel blocker nifedipine (3 μM) or Ca2+-free solution. Nifedipine did not affect the methacholine-induced catecholamine responses, but it inhibited the responses during treatment with apamin. The L-type Ca2+channel activator Bay k 8644 (1 μM) enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced epinephrine and norepinephrine responses were prevented and attenuated by apamin, respectively. These results suggest that SKCachannels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca2+channels and thereby attenuates adrenal catecholamine secretion.

To investigate the effect of angiotensin (ANG) II on aldosterone (ALDO) secretion, we measured arterial and adrenal venous plasma aldosterone concentrations in anesthetized dogs. The intraadrenal arterial infusion of ANG II (0.3 ng/kg/min) or potassium chloride (KCl) (0.6 mg/min) increased ALDO secretion. The changes in ALDO secretion in response to ANG II were tested during the concomitant arterial infusion of two graded doses of losartan (10 and 100 ng/kg/min), PD 123319 (50 and 500 ng/kg/min), nifedipine (25 and 250 ng/kg/min), or TMB-8 (2 and 20 microg/kg/min). All of these test drugs except PD123319 inhibited the ANG II-induced increase in ALDO secretion. Losartan did not affect the KCl-induced increase in ALDO secretion. These results indicate that ANG II acts on ANG II type 1 receptors in the adrenal gland and enhances ALDO secretion. They also suggest the involvement of both intracellular and extracellular calcium in the aldosterone response to stimulation by ANG II. Under these in vivo experimental conditions, the KCl-stimulated ALDO secretion does not appear to involve ANG II formation in the adrenal gland.

We examined the participation of endothelin ET A and ET B receptors in modulation by endothelin-1 of adrenal catecholamine secretion during cholinergic activation in pentobarbital-anesthetized dogs. Drugs were infused intra-arterially into the adrenal gland. Splanchnic nerve stimulation (1 and 3 Hz) increased adrenal catecholamine output in a frequency-dependent manner. Endothelin-1 (0.2, 0.6, and 2 ng/kg/min) enhanced the catecholamine response induced by the 3-Hz nerve stimulation. Under pretreatment with an endothelin ET A receptor antagonist ( R )-2-[( R )-2-[( S )-2-[[1-(hexahydro-1 H -azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-(2-pyridyl) propionic acid (FR139317) (1 μg/kg/min), endothelin-1 suppressed the 1- and 3- Hz nerve stimulation-induced catecholamine response in a dose-dependent manner. No inhibitory or facilitatory effect of endothelin-1 was observed under simultaneous pretreatment with FR139317 and an endothelin ET B receptor antagonist N - cis 2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine (BQ-788) (1 μg/kg/min) or under pretreatment with BQ-788 alone. These results suggest that in the dog adrenal gland, endothelin-1 facilitates and inhibits adrenal catecholamine secretion during cholinergic activation by stimulating endothelin ET A and ET B receptors, respectively.

We evaluated the effects of an adenylate cyclase activator, N, N-dimetyl-beta-alanine[3R-(3alpha,4alphabeta,5beta+ ++,6beta,6aalpha, 10alpha,10abeta,10balpha)]-5(acetyloxy)-3-eth enyldodecahydro-10, 10b-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-1H-naphtho[2, 1-b]pyran-6-yl ester hydrochloride (NKH477), on neural control of renal functions in anesthetized dogs. Renal nerve stimulation (2 Hz) increased renal norepinephrine efflux and reduced renal blood flow, glomerular filtration rate, urine flow rate, urinary Na(+) excretion and fractional Na(+) excretion. Intrarenal arterial infusion of NKH477 (300 ng/kg/min) suppressed the stimulation-induced reductions in renal blood flow and glomerular filtration rate and attenuated the reductions in urine flow rate and urinary Na(+) excretion but not the changes in renal norepinephrine efflux and fractional Na(+) excretion. Infusion of NKH477 did not affect the urinary responses induced by renal nerve stimulation at a lower frequency (0.5-1 Hz) which had little influence on renal blood flow and glomerular filtration rate. The present results demonstrate that NKH477 inhibits renal vasoconstriction and hypofiltration but not the enhanced tubular Na(+) reabsorption during activation of the renal sympathetic nervous system.

We examined effects of gamma-aminobutyric acid (GABA) on vasoconstriction and noradrenaline (NA) release induced by electrical renal nerve stimulation (RNS) in the isolated pump-perfused rat kidney. RNS (1 and 2 Hz for 2.5 min each, 0.5-ms duration, supramaximal voltage) increased renal perfusion pressure (PP) and renal NA efflux. GABA (3, 10 and 100 microM) attenuated the RNS-induced increases in PP by 10-40% (P

Although the renal nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) system plays an important role in maintaining urinary sodium and water excretion, effects of an authentic NO donor sodium nitroprusside (SNP) on urine formation have been controversial. In this study, we examined whether SNP increases renal NO release and cGMP production and induces natriuresis in the denervated kidney of anesthetized dogs. The intrarenal arterial infusion of SNP at 10, 30, and 100 ng/kg/min did not affect renal function or NO-cGMP production. The higher dose of SNP (1.000 ng/kg/min) reduced systemic blood pressure and urine flow rate. The antidiuresis was observed also in the contralateral control kidney, the degree of which was larger than that observed in the ipsilateral SNP-infused kidney. During the SNP infusion, reductions in urinary Na + excretion, fractional Na + excretion. and urinary nitrite + nitrate excretion occurred in the control kidney but not in the SNP-infused kidney. Urinary cGMP excretion and renal venous plasma cGMP concentration were significantly increased during the SNP infusion in the SNP-infused kidney but not in the control kidney. These renal effects of SNP were similar to those obtained by intrarenal arterial infusion of a specific NO donor, NOC 7 (300 ng/kg/min). These results suggest that SNP can produce nitric oxide and increase cGMP levels in the kidney and suppress sodium reabsorption. but the natriuretic property of SNP may be masked by its counteracting effects including the systemic hypotension in anesthetized dogs.

IL-1 and TNF-alpha are known to be pleiotropic cytokines associated with various inflammatory conditions such as small intestinal injury after ischemia-reperfusion. FR167653 has been characterized as a potent suppressant of IL-1 and TNF-alpha production. The effect of FR167653 on intestinal reperfusion injury was investigated in a warm ischemia model of the canine gut. Sixteen mongrel dogs were divided into two groups: a control group and a FR group to which FR167653 was administered. Both the superior mesenteric artery and vein were clamped for 2 hr. Arterial pH, hepatic venous hemoglobin oxygen saturation, intramucosal pH, and the survival rate were well maintained in the FR group in comparison with the control group after reperfusion. FR167653 inhibited the expression of IL-1beta mRNA. Histologically, ischemia-reperfusion injury was more severe in the control group than the FR group. This study suggests that FR167653 inhibits proinflammatory cytokines and ameliorates ischemia-reperfusion injury of the small intestine.

SUMMARY 1. We examined whether zaprinast, a putative cGMP-specific phosphodiesterase inhibitor, affects neural control of renal function in pentobarbital-anaesthetized dogs. 2. Renal nerve stimulation (1Hz, 1ms duration) reduced urine flow rate, urinary Na+ excretion (UNaV) and fractional excretion of Na+ (FENa) with little change in either renal blood flow (RBF) or glomerular filtration rate (GFR). 3. Intrarenal arterial infusion of zaprinast (10 and 100 μg/kg per min) increased basal urine flow rate, UNaV and FENa but not RBF or GFR. Zaprinast infusion (100 μg/kg per min) also increased renal venous plasma cGMP concentration and urinary cGMP excretion. 4. Renal nerve stimulation-induced reductions in UNaV and FENa were attenuated during zaprinast infusion, whereas the reduction in urine flow rate was resistant to zaprinast. 5. Renal nerve stimulation increased the renal venous plasma noradrenaline concentration and renal noradrenaline efflux, which remained unaffected during infusion of zaprinast (100 μg/kg per min). 6. The results of the present study suggest that zaprinast induces natriuresis and counteracts adrenergically induced anti-natriuresis by acting on renal tubular sites in the dog kidney in vivo.

We investigated the effect of cilnidipine, a novel dihydropyridine Ca2+ channel blocker possessing blocking actions on N-type and L-type voltage-dependent Ca2+ channels (VDCCs), in comparison with the L-type VDCC blocker nifedipine, on adrenal catecholamine secretion in response to splanchnic nerve stimulation (SNS), acetylcholine (ACh), the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and muscarine in anesthetized dogs. Ca2+ channel blockers and cholinergic agonists were infused and injected, respectively, into the adrenal gland through the phrenicoabdominal artery. Cilnidipine (0.3-3 microg/min) inhibited increases in both epinephrine (EPI) and norepinephrine (NE) output induced by SNS (2 Hz), ACh (1.5 microg), and DMPP (0.2 microg). However, cilnidipine inhibited increase in NE output induced by muscarine (1 microg) without affecting increase in EPI output. Nifedipine (0.3-3 microg/min) inhibited the ACh- and DMPP-induced increases in EPI and NE output without affecting the SNS- and muscarine-induced increases in EPI and NE output. From these results, it seems likely that the inhibition by cilnidipine of the SNS-induced EPI and NE secretion and of the muscarine-induced NE secretion is related to its blocking action on N-type VDCCs.

Intrarenal arterial infusion of norepinephrine (30 ng/kg per min) or of angiotensin II (4 ng/kg per min) reduced the glomerular filtration rate and urinary Na + excretion in denervated kidneys of anesthetized rabbits pretreated intrarenally with a nitric oxide (NO) synthase inhibitor N ω -nitro- l -arginine methyl ester (50 μ g/kg per min). Angiotensin II but not norepinephrine reduced fractional Na + excretion. Intrarenal administration of a spontaneous NO donor 1-hydroxy-2-oxo-3-( N -methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7, 30 ng/kg per min) in l -NAME pretreated kidneys did not affect basal values, but attenuated the reduction in urinary Na + excretion induced by these agonists without affecting the angiotensin II-induced reduction in glomerular filtration rate. The results suggest that NOC 7 can suppress the norepinephrine-induced hypofiltration and the angiotensin II-evoked tubular reabsorption and thereby attenuates the agonist-induced antinatriuresis in the denervated and endogenous NO-depleted rabbit kidney.

The effects of NKH477, a water-soluble forskolin derivative, and dibutyryl-cyclic adenosine monophosphate (dbcAMP) on the release of adrenal catecholamines (CAs) in response to splanchnic nerve stimulation (SNS), acetylcholine (ACh), the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) and muscarine were examined in anesthetized dogs. NKH477, dbcAMP and the cholinergic agonists were infused and injected, respectively, into the adrenal gland intra-arterially. SNS (3 Hz) or injections of ACh (3 micrograms), DMPP (2 micrograms) and muscarine (2 micrograms) produced increases in CA output determined from adrenal venous blood. Both NKH477 infusion (0.3, 1 and 3 micrograms/min) and dbcAMP infusion (0.1, 0.3 and 1 mg/min) caused dose-dependent enhancement of the SNS-, ACh- and DMPP-induced increases in CA output, whereas they failed to affect the muscarine-induced increases in CA output. Neither NKH477 nor dbcAMP affected basal CA output. Cyclic AMP (cAMP) overflow determined from adrenal venous blood increased during NKH477 infusion. These results indicate that NKH477 and dbcAMP have facilitatory effects on adrenal CA release mediated by nicotinic receptors, but not by muscarinic receptors in the dog, and suggest the selective action of cAMP on nicotinic mechanism.

Intrarenal arterial infusion of C-type natriuretic peptide (CNP, 50 ng/kg per min) increased urine flow rate without affecting glomerular filtration rate. Intrarenal arterial bolus injection of angiotensin II (25, 50 and 100 ng) or of norepinephrine (0.25, 0.5 and 1.0 microg) reduced renal blood flow. The blood flow response induced by angiotensin II was slightly attenuated but the response induced by norepinephrine was unaffected during CNP infusion. These results suggest that exogenous CNP, even at the pharmacological dose that can induce diuresis, has little effect on the canine renal vasculature.

Multiple cholesterol stones are associated with more biliary complications and show more rapid cholesterol nucleation than solitary stones. Group II phospholipase A2 (PLA2-II) may play a critical role in the process of mucosal inflammation, which in turn may produce pronucleating agents. PLA2-II concentrations in gallbladders and gallbladder bile from patients with different types of gallstone disease were assayed to correlate PLA2-II with alterations in biliary composition.PLA2-II protein concentrations were assayed immunoradiometrically using monoclonal antibodies against human splenic PLA2-II.Immunoreactive PLA2-II levels in gallbladder bile were significantly higher in patients with multiple cholesterol stones (68.2 +/- 6.3 ng/dL, mean +/- SEM; n = 24) than in those with solitary stones (24.9 +/- 2.8; n = 20; P0.01), those with multiple pigment stones (24.2 +/- 3.7; n = 18; P0.01), or control subjects (13.4 +/- 1.7; n = 19; P0.01). Increased biliary immunoreactive PLA2-II levels in multiple cholesterol stones were associated with a concomitant increase in the lysophosphatidylcholine to phosphatidylcholine ratio; free arachidonate, protein, and hexosamine concentrations; and gallbladder bile viscosity. The gallbladders showed an increased PLA2-II protein mass and steady-state messenger RNA levels, which was associated with increased prostaglandin E2 levels.Increased biliary PLA2-II may be of pathogenetic importance in multiple cholesterol stones, probably through potentiating gallbladder mucosal inflammation with associated biliary alterations favoring cholesterol crystal formation.

We investigated the role of high conductance (BK(Ca)) and small conductance Ca2(+)-activated K+ (SK(Ca)) channels in adrenal catecholamine release in response to splanchnic nerve stimulation, acetylcholine, the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and muscarine in anesthetized dogs. The selective SK(Ca) channel blocker apamin and the selective BK(Ca) channel blocker charybdotoxin were infused into the adrenal gland through the phrenicoabdominal artery, and the cholinergic agonists were injected into the same artery. Splanchnic nerve stimulation (1, 2, 3 and 10 Hz), acetylcholine (0.75, 1.5 and 3 microg), DMPP (0.1, 0.2 and 0.4 microg) and muscarine (0.5, 1 and 2 microg) produced frequency- or dose-dependent increases in catecholamine output as measured in adrenal venous blood. Apamin infusion (1, 3 and 10 ng/min) enhanced the acetylcholine-, DMPP- and muscarine-induced increases in catecholamine output in a dose-dependent manner, but it did not affect the splanchnic nerve stimulation-induced catecholamine response. Charybdotoxin infusion (10, 30 and 100 ng/min) did not affect the increases in catecholamine output induced by the agonists and splanchnic nerve stimulation. Neither apamin nor charybdotoxin affected basal catecholamine output. These results suggest that apamin-sensitive SK(Ca) channels located in adrenal medullary cells may play an inhibitory role in the regulation of adrenal catecholamine release mediated by extrasynaptic nicotinic and muscarinic receptors.

We examined the effects of glucagon on the release of adrenal catecholamines (CAs) in response to splanchnic nerve stimulation (SNS), acetylcholine (ACh), the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), and muscarine in anesthetized dogs. Glucagon and the cholinergic drugs were infused and injected, respectively, into the adrenal gland through the phrenicoabdominal artery. SNS (1 and 3 Hz) or injections of ACh (1.5 and 3 micrograms), DMPP (1 and 2 micrograms), and muscarine (1 and 2 micrograms) produced a frequency- or dose-dependent increase in both epinephrine (EPI) and norepinephrine (NE) output, determined from adrenal venous blood. Glucagon infusion (0.1, 0.3, and 1 microgram/min) enhanced the SNS-, ACh-, and DMPP-induced increases in EPI and NE output in a dose-dependent manner but did not affect the muscarine-induced increases in CA output. The increase in basal CA output induced by the highest dose of glucagon was only slight. Glucagon increased cyclic AMP overflow determined from adrenal venous blood. Our results indicate that glucagon has a facilitatory action on adrenal CA release mediated by nicotinic receptors but not by muscarinic receptors in dogs and suggest that an increase in cyclic AMP production in adrenal medullary cells may be responsible for its selective action.

1. The aim of this study was to investigate the renal vascular response to angiotensin II (3-8) (AIV). The effect of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) or the cyclooxygenase inhibitor, indomethacin on the AIV-induced response was examined in anaesthetized spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). 2. Intrarenal infusion of AIV produced a biphasic vasoconstrictor response. The vasoconstriction developed rapidly to reach a maximum followed by a partial recovery to a sustained lesser level of vasoconstriction. The initial maximum response was enhanced by L-NAME but not affected by indomethacin treatment. The simultaneous administration of L-NAME and indomethacin prevented the partial recovery resulting in a greater sustained level of constriction. 3. A stable vasoconstriction of relatively constant magnitude was observed with angiotensin II (AII) infusion. The AII vasoconstriction was enhanced by L-NAME but not changed by indomethacin. The combination of these inhibitors further enhanced the AII-induced vasoconstriction in WKY, but not in SHR. 4. Pretreatment with the AII receptor antagonist, losartan, inhibited the vasoconstriction induced by AIV and AII. 5. These results suggest that nitric oxide and prostaglandins may modulate the renal vascular response to AIV.

A method to produce a mecasermin fused protein on an industrial scale was investigated. We constructed a new expression vector (designated pLSD1) derived from pLHSdMmtrp (Saito, Y. et al.: J. Biochem., 101, 123–134, 1987) by introduction of a synthetic fd phage terminator and deletion of the rop region originating from pBR322. The plasmid, pLSD1, exhibited high stability and was present at high copy number in Escherichia coli HB101. Although the growth of E. coli HB101 pLSD1 was not sufficient for high-level production of the fused protein in a Trp-deficient medium such as M9CA medium, it was improved by growing the strain in a medium containing 0.7% yeast extract which was constantly supplied with glucose. E. coli HB101 requires Pro and Leu for its growth; however, excess Leu tended to inhibit the cell growth. From the results of investigation of the mechanism of inhibition by Leu, addition of Ile was found to prevent the inhibition. Consequently, high-level production of the fused protein was attained by (i) using pLSD1 as the expression vector, (ii) cultivation of E. coli HB101 pLSD1 in a medium containing 0.7% yeast extract, (iii) fed-batch cultivation with periodic addition of glucose, and (iv) addition of Pro, Leu and Ile during cultivation. The cell density reached an OD at 600 nm of 50.4 and production of the fused protein was 1.24 g/l broth in a 150 l fermentor.

Recently, soluble(s) circulating isoforms of intercellular adhesion molecule-1 (sICAM-1) and sE-selectin (formerly endothelial leukocyte adhesion molecule-1) have been described in normal human serum. Elevated levels have been reported in acute and chronic inflammatory disorders, including allograft rejection. In this study, plasma levels of sICAM-1 and sE-selectin were determined in groups of tacrolimus (FK 506)-treated adult patients following either isolated small bowel (SB), liver, or combined SB plus liver (SB/L) transplantation. Each molecule was measured at 1, 2, 4, 6, 8, and 12 weeks (all patients) and at 6, 9, and 12 months after transplantation (SB and SB/L only) by enzyme linked immunosorbent assay. Levels were compared with those of soluble interleukin-2 receptor (sIL-2R ; a marker of lymphocyte activation) and soluble HLA class I (which has been reported to be elevated in liver transplant-related complications). Elevations above normal in mean plasma levels of sICAM-1 (2.4-fold), sE-selectin (1.8-fold), sIL-2R (10.6-fold), and sHLA class I (1.3-fold) were found in patients with stable isolated SB grafts during the first 12 weeks posttransplant. Except for sHLA class I, levels of each protein were subsequently reduced, up to 1 year posttransplant. However, further increases in sICAM-1 and in sIL-2R and sE-selectin levels were observed during episodes of SB rejection compared with stable grafts. Mean levels of all molecules were higher in patients with isolated SB grafts compared with those given liver or combined (SB/L) transplants, either during stable SB graft function (up to 12 weeks posttransplant) or rejection. The data demonstrate increased adhesion molecule production/shedding following SB transplantation and are suggestive of a reduced overall level of immune activation in liver and SB/L compared with isolated SB transplantation.

1. To clarify the possible role of tissue catecholamines in the development of hypertension, we investigated the effect of bilateral renal denervation on the catecholamine contents of central and peripheral tissues in spontaneously hypertensive rats (SHR). 2. Norepinephrine (NE) content in renal cortex, renal medulla, and adrenal gland was higher in 7 week old SHR than age-matched Wistar-Kyoto rats (WKY). Dopamine (DA) content in the brainstem and hypothalamus was also higher in SHR, but NE and epinephrine (EPI) content in these areas were not different between strains. Similar differences in catecholamines were observed in 9 week old rats in which a sham operation of bilateral renal denervation was performed 2 weeks previously. 3. Bilateral renal denervation produced an almost complete reduction of NE content in the kidney in both strains and prevented the development of hypertension. DA content in the brainstem was also decreased by renal denervation in SHR but not in WKY. NE and EPI content in central tissues were not affected by renal denervation. 4. These results suggest that DA content in brainstem area, as well as NE content in the kidney, have a relationship in the development of hypertension in SHR.

The effects of yohimbine and desipramine on adrenal catecholamine (CA) release in response to splanchnic nerve stimulation (SNS) were examined in anesthetized dogs. SNS and 3 Hz produced frequency-dependent increases in epinephrine (EPI) and norepinephrine (NE) output determined from adrenal venous blood. Yohimbine (30 and 100 micrograms/kg, i.v.), a selective alpha 2-adrenoceptor antagonist, enhanced the SNS-induced increases in both EPI and NE output. Desipramine (100 and 300 micrograms/kg, i.v.), an amine pump inhibitor, enhanced the SNS-induced increases in NE output, whereas no enhancement of EPI output was produced. After desipramine treatment, yohimbine further enhanced the SNS-induced increases in EPI and NE output. After yohimbine treatment, desipramine further enhanced the SNS-induced increase in NE output. These results suggest that the release of adrenal CA in response to SNS is inhibited by alpha 2-adrenoceptors, and that released NE, rather than EPI, is predominantly taken up into the dog adrenal medullary cells.

Cranioplasty with autogenous split-thickness calvarial bone was performed in 10 patients. Follow-up for a mean of 19 months (range 2-43 mos) showed satisfactory protection of the brain and cosmetic reconstruction. No serious complication was seen except in one patient with postoperative epidural abscess. Split-thickness calvarial bone graft is recommended in patients with previous infection or high risk of infection, in poorly vascularized recipient sites resulting from multiple operations or irradiation, and in younger patients aged more than 7 years.

This study investigates the molecular mechanisms underlying the induction of and protection from T cell activation-associated hepatic injury. When BALB/c mice were given a single intravenous injection of concanavalin A (Con A) (> or = 0.3 mg/mouse), they developed acute hepatic injury as assessed by a striking increase in plasma transaminase levels within 24 h. Histopathologically, only the liver was injured while moderate infiltration of T cells and polymorphonuclear cells occurred in the portal areas and around the central veins. The induction of hepatic injury was dependent on the existence as well as the activation of T cells, as untreated BALB/c nu/nu mice or BALB/c mice pretreated with a T cell-specific immunosuppressive drug, FK506, failed to develop disease. Significant increases in the levels of various cytokines in the plasma were detected before an increase in plasma transaminase levels. Within 1 h after Con A injection, tumor necrosis factor (TNF) levels peaked, this being followed by production of two other inflammatory cytokines, interleukin 6 (IL-6) and IL-1. Passive immunization with anti-TNF but not with anti-IL-1 or anti-IL-6 antibody, conferred significant levels of protection. Moreover, administration of rIL-6 before Con A injection resulted in an IL-6 dose-dependent protection. A single administration of a given dose of rIL-6 completely inhibited the release of transaminases, whereas the same regimen induced only 40-50% inhibition of TNF production. More than 80% inhibition of TNF production required four consecutive rIL-6 injections. These results indicate that: (a) TNFs are critical cytokines for inducing T cell activation-associated (Con A-induced) hepatitis; (b) the induction of hepatitis is almost completely controlled by rIL-6; and (c) rIL-6 exerts its protective effect through multiple mechanisms including the reduction of TNF production.

The induction of macrophage colony-stimulating factor (M-CSF) in monkey plasma following administration of FK565 was observed within 2 h of injection peaked at 4 h, and remained high after 24 h. Interleukin-6 (IL-6) and M-CSF levels increased in monkeys treated with FK565, even at doses as low as 0.01 mg/kg. Granulocyte CSF (G-CSF) levels increased slightly following a dose of 1 mg/kg, but granulocyte macrophage CSF (GM-CSF) was not detected at any doses of FK565 studied. To examine the thrombopoietic activity of FK565 in vivo, single doses of drug (0.01, 0.1 or 1.0 mg/kg) were administered i.v. to cynomolgus monkeys or normal mice on day 0. The promotes platelet (PLT) count after FK565 injection decreased transiently on days 1 and 2, and then increased in a dose-dependent manner on day 5 and was still high on day 14. The experiment using anti-PLT antibody showed that the increased PLT count was not simply due to a rebound phenomenon after the transient decrease in PLT. The effect of i.v. FK565 was studied in mice myelosuppressed with a single dose of mitomycin C (MMC) (5.6 mg/kg). The fall in PLT count was suppressed on day 7 by 0.1 and 1.0 mg/kg FK565. Although intact cells or tissues are necessary for an increase in PLT following FK565 treatment, FK565 suppressed the impaired hematopoietic function seen after chemotherapy. FK565 is proposed as a drug to restore reduced neutrophil and platelet counts found in AIDS or cancer therapy.

山形市の破裂脳動脈瘤患者の発生状況を調べるため, 1987～1989年の3年間に発症した市内在住者の破裂脳動脈瘤を地域内の全脳神経外科5施設の協力のもとに集計した.症例は脳血管写または手術により診断された123例と臨床経過ならびにCTにより診断された25例の計148例であった.発生率は19.9人/10万人/年であった.年代別の症例数は50歳台, 70歳台が共に36例と最多であったが, 発生率の最多は70歳台であった (82.2人/10万人/年).患者の年齢は23～85歳で平均58.9歳, 男性56例, 女性92例であった.70歳以上の症例では女性が男性の2.7倍と性差が著明であった.入院時grade (Hunt&Kosnik分類, 付帯事項除外) はI・II62例 (41.8%), III32例 (21.6%), IV13例 (8.8%), V41例 (27.7%), 来院時心肺停止症例は14例 (全体の9.4%) であった.今回の調査において年間発生率, 入院時最重症例の比率は他の地域で報告されている値に比べ, 高値であった.

We investigated the way in which hexamethonium and pirenzepine, a selective muscarinic (M) 1 receptor antagonist, modify the release of catecholamines from dog adrenal gland in response to endogenous and exogenous acetylcholine (ACh) in vivo. Output of epinephrine (EPI), and norepinephrine (NE) was determined from adrenal venous blood by high-performance liquid chromatography (HPLC) with electrochemical detection. Intraarterial (i.a.) injections of ACh (1.5 and 3 micrograms) or muscarine (3 and 6 micrograms) into the phrenicoabdominal artery and splanchnic nerve stimulation (SNS, 3 Hz) produced marked increases in EPI and NE output. Hexamethonium (1 mg/kg intravenously, i.v.) inhibited the increases in EPI and NE output in response to exogenous ACh and SNS. Pirenzepine (10 micrograms/kg i.v.) inhibited the ACh-induced increases in EPI and NE output but did not modify the SNS-induced increases. The muscarine-induced increases in EPI and NE output were also inhibited by pirenzepine. These results indicate that the exogenous ACh-induced release of adrenal catecholamines is mediated through both nicotinic and M1 receptors, in contrast to the predominant mediation of nicotinic receptors in response to endogenous ACh.

Effects of the renin-angiotension system (RAS) on adrenal catecholamine release in response to hemorrhagic hypotension and splanchnic nerve stimulation (SNS) were studied in pentobarbital-anesthetized dogs. In hemorrhage experiments, mean blood pressure (MBP) was maintained at 50 mm Hg for 60 min by bleeding the arterial blood into a pressurized bottle. In the renal intact group (control), epinephrine (EPI) and norepinephrine (NE) output from the adrenal gland increased markedly during hemorrhagic hypotension: from 45 +/- 13 and 4.7 +/- 0.9 to 1,167 +/- 202 and 169 +/- 30 ng/min at 60 min after onset of hemorrhage, respectively. The increases in catecholamine output during hemorrhagic hypotension in the renal-intact group pretreated with captopril (1 mg/kg intravenously, i.v.) and in the renal-ligated group were significantly smaller than those in the control group. The increases in catecholamine output in the renal-ligated group infused with angiotensin II (AngII 10 ng/kg/min i.v.) were comparable to those in the control group. In SNS experiments, AngII infusion (10 ng/kg/min i.v.) enhanced increases in catecholamine output induced by 3 Hz SNS significantly. Captopril (1 mg/kg i.v.) did not affect the SNS-induced increases in catecholamine output. These results suggest that the renal RAS facilitates reflex release of adrenal catecholamines during hemorrhagic hypotension, at least in part, by acting directly on the release process of catecholamines from dog adrenal gland.

Serotonin (5-HT 0.1 and 0.3 micrograms/kg) was injected as a bolus into the renal artery of anesthetized dogs. Renal blood flow (RBF) decreased initially after 5-HT injection and then increased. The 5-HT-induced decrease in RBF was potentiated during intrarenal infusion of a 5-HT1 and 5-HT2 antagonist methysergide at 3 micrograms/kg/min or of a 5-HT2 antagonist ketanserin (3-30 micrograms/kg/min), but methysergide at 30 micrograms/kg/min attenuated the decrease in RBF. The delayed increase in RBF was suppressed during ketanserin or methysergide infusion. In ketanserin-pretreated dogs, methysergide (3-30 micrograms/kg/min) dose-dependently suppressed the 5-HT-induced decrease in RBF. A 5-HT3 antagonist, ICS 205-930 (3-30 micrograms/kg/min), did not affect the 5-HT-induced RBF responses. A Ca2+ entry blocker CD-349 (30 and 100 ng/kg/min) and a Ca2+ release inhibitor TMB-8 (30 and 100 micrograms/kg/min) suppressed the 5-HT-induced decrease in RBF. These results suggest that 5-HT activates 5-HT1 receptors to induce vasoconstriction by mobilization of extracellular and intracellular Ca2+, but simultaneous activation of 5-HT2 receptors attenuates the vasoconstriction, probably by causing vasodilation in dog kidney.

The effects of dopamine (DA)-receptor and alpha 2-adrenoceptor agonists and antagonists on ganglionic muscarinic stimulation were examined in anesthetized dogs. All drugs were injected or infused intra-arterially into the blood supply of the cardiac sympathetic ganglia. The muscarinic agonists McN-A-343 (10, 20, and 30 micrograms) and muscarine (1, 2, and 3 micrograms) increased heart rate. The muscarinic receptor stimulation elicited by McN-A-343 or muscarine was significantly inhibited by infusion of the DA2-receptor agonist quinpirole (3 and 10 micrograms/min) but not by the DA1-receptor agonist SK&F 38393 (10 and 30 micrograms/min). The alpha 2-adrenoceptor agonist BHT 933 (3 and 10 micrograms/min) also inhibited muscarinic receptor stimulation. The DA2-receptor antagonist domperidone (10 micrograms/min) and the alpha 2-adrenoceptor antagonist yohimbine (1 micrograms/min) had no effects on muscarinic receptor stimulation, but they antagonized the inhibitory effects of quinpirole and BHT 933, respectively. The nicotinic transmission elicited by preganglionic cardiac sympathetic nerve stimulation (1 and 2 Hz) was also inhibited by DA-receptor and alpha 2-adrenoceptor agonists and antagonists. These results suggest that DA2-receptors and alpha 2-adrenoceptors suppress muscarinic transmission as well as nicotinic transmission in the cardiac sympathetic ganglia of the dog.

SUMMARY 1. Intrarenal arterial infusion of a direct adenylate cyclase activator (NKH477; 300 ng/kg per min) increased renal blood flow, urine flow rate and urinary sodium excretion in anaesthetized dogs. 2. Intrarenal arterial infusion of endothelin (ET)-1 (2 ng/kg per min) reduced basal values of these parameters and glomerular filtration rate, which were recovered by the addition of NKH477 during ET-1 infusion. 3. These results demonstrate that NKH477 can counteract ET-1-induced antinatriuresis, mainly by restoring glomerular filtration.

In an investigation of (-)-Cercosporamide derivatives with a plasma glucose-lowering effect, we found that N-benzylcarboxamide derivative 4 was a partial agonist of PPARgamma. A SAR study of the substituents on carboxamide nitrogen afforded the N-(1-naphthyl)methylcarboxamide derivative 23 as the most potent selective PPARgamma modulator. An X-ray crystallography study revealed that compound 23 bounded to the PPARgamma ligand binding domain in a unique way without any interaction with helix12. Compound 23 displayed a potent plasma glucose-lowering effect in db/db mice without the undesirable increase in body fluid and heart weight that is typically observed when PPARgamma full agonists are administrated.