Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up-regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin-1 receptor antagonist-deficient mice.

BACKGROUND AND OBJECTIVE: The interleukin (IL)-1 receptor antagonist (Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is unclear whether the IL-1Ra plays a protective role in periodontal disease. The purpose of this study was to compare IL-1Ra knockout (KO) and wild-type (WT) mice in regard to proinflammatory cytokine production, osteoclast formation and bone resorption in response to periodontal bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Peritoneal macrophages (Mφs) were obtained from 13-wk-old IL-1Ra KO and WT mice. Peritoneal Mφs were cultured with or without 10 µg/mL of Aggregatibacter actinomycetemcomitans LPS for 24 h. The levels of IL-1alpha (IL-1α), IL-1beta (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 were measured in periotoneal Mφs supernatant fluid (PM-SF) using an ELISA. Bone marrow cells were obtained from the mice and stimulated with PM-SF for 9 d, then stained with TRAP. The frequency of TRAP-positive multinucleated giant cell formation was calculated based on a fusion index. PM-SF-stimulated calvarial bone resorption was analyzed using micro-computed tomography, and calvarial histological analysis was performed using hematoxylin and eosin and TRAP staining. The expression of cyclooxygenase-2 (Cox2), prostanoid receptor EP4 (Ep4) and Rank mRNAs in bone marrow cells were measured using real-time quantitative PCR, while prostaglandin E2 (PGE2 ) production was determined by ELISA. RESULTS: The levels of IL-1α, IL-1ß, TNF-α and IL-6 in IL-1Ra KO mice PM-SF stimulated with A. actinomycetemcomitans LPS were significantly increased by approximately 4- (p < 0.05), 5- (p < 0.05), 1.3- (p < 0.05) and 6- (p < 0.05) fold, respectively, compared with the levels in WT mice. Moreover, osteoclast formation, expression of Rank, Ep4 and Cox2 mRNAs and production of PGE2 were significantly increased by approximately 2- (p < 0.05), 1.6- (p < 0.05), 2.5- (p < 0.05), 1.6- (p < 0.05) and 1.9- (p < 0.05) fold, respectively, in IL-1Ra KO mice stimulated with A. actinomycetemcomitans LPS compared with WT mice. CONCLUSION: IL-1Ra regulates IL-1 activity and appears to reduce the levels of other inflammatory cytokines, including TNF-α and IL-6, while it also reduces expression of the EP4 receptor related to prostanoid sensitivity and osteoclast formation. These results suggest that IL-1Ra is an important molecule for inhibition of inflammatory periodontal bone resorption.

Free radical mediation of the effects of acidosis on calcium transport by cardiac sarcoplasmic reticulum in whole heart homogenates.

Generation of oxygen free radicals by xanthine acting on xanthine oxidase as a substrate significantly depressed calcium transport by sarcoplasmic reticulum in canine whole heart homogenates at 37 degrees C. At pH 7.0, this effect was completely inhibited by the addition of superoxide dismutase (SOD), a scavenger of the superoxide anion radical. At pH 6.4, SOD (5 to 20 micrograms X ml-1) was ineffective but catalase (20 micrograms X ml-1) was able to inhibit the effects of the xanthine-xanthine oxidase system. SOD + catalase (20 micrograms X ml-1) and SOD + mannitol, a scavenger of the hydroxyl free radical, inhibited the effects of the xanthine-xanthine oxidase system at pH 6.4. Preincubation at pH 6.4, in the absence of an exogenous free radical generating system, depressed calcium transport. This depression was more severe the longer the duration of incubation. However, return of the pH to 7.0 after preincubation at pH 6.4 partially restored calcium uptake velocity. The degree of reversibility was decreased the longer the period of incubation at pH 6.4. SOD reversed the effects of incubation at pH 6.4 for 5 min, but not those for incubations of 10 and 15 min. Mannitol alone was ineffective. The combinations of SOD and mannitol significantly reversed the effects of pH 6.4 up to 15 min. These results demonstrate that both exogenously generated and endogenously generated free oxygen radicals are capable of depressing calcium transport by cardiac sarcoplasmic reticulum in the whole heart homogenate in the presence of endogenous scavenging systems.(ABSTRACT TRUNCATED AT 250 WORDS)

The cleavage site specificity of human prostate specific antigen for insulin-like growth factor binding protein-3.

The cleavage site of human insulin-like growth factor binding protein-3 by urinary prostate specific antigen was examined. Human insulin-like growth factor binding protein-3 was incubated with urinary prostate specific antigen at 37 degrees C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N-terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr-159. The synthetic peptide including Tyr-159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin-like growth factor binding protein-3 is specifically cleaved at Tyr-159 by prostate specific antigen. Human insulin-like growth factor binding protein-3 was previously reported to be cleaved at five sites including Arg-97, Arg-132, Tyr-159, Phe-173 and Arg-179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin-like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin-like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin-like growth factor binding protein-3.

Calmodulin and cyclic ADP-ribose interaction in Ca2+ signaling related to cardiac sarcoplasmic reticulum: superoxide anion radical-triggered Ca2+ release.

Reactive oxygen species (ROS) are often shown to damage cellular functions. The targets of oxidative damage depend on the nature of ROS produced and the site of generation. In contrast, ROS can also regulate signal transduction. In this case, ROS may either induce or enhance events, which lead to forward directions of cellular signaling. The consequences of regulation of signal transduction can be observed in physiological processes such as muscle contraction. Here, we discuss the concentration-dependent effects of superoxide anion radical (*O2-) on Ca2+ release from the cardiac sarcoplasmic reticulum (SR). Recent studies suggest that the ADP-ribosyl cyclase pathway, through its production of cyclic adenosine 5'-diphosphoribose (cADPR), may control Ca2+ mobilization in cardiac muscle cells. *O2- has dual effects that are concentration dependent. At low concentrations (nearly nanomolar levels), *O2- induces Ca2+ release by stimulating synthesis of cADPR, which requires calmodulin for sensitization of ryanodine-sensitive Ca2+-release channels (RyRC). At these low concentrations, *O2- is responsible for regulation of cellular signal transduction. At higher concentrations (micromolar levels), *O2- produces a loss in the function of calmodulin that is to inhibit RyRC. This results in an increase in Ca2+ release, which is linked to cell injury. The difference in the functions of low and high concentrations of *O2- may result in two distinct physiological roles in cardiac muscle Ca2+ signaling.

Novel mechanisms involved in superoxide anion radical-triggered Ca2+ release from cardiac sarcoplasmic reticulum linked to cyclic ADP-ribose stimulation.

It has been suggested that cyclic adenosine 5'-diphosphoribose (cADPR) directly activates the cardiac isoform of the ryanodine receptor (RyR)/Ca2+ release channel. We have previously shown that selective activation of RyR/Ca2+ release channel by superoxide anion radical (O2.-) is dependent of the presence of calmodulin and identified calmodulin as a functional mediator of O2.- -triggered Ca2+ release through the RyR/Ca2+ release channel of cardiac sarcoplasmic reticulum (SR). We now demonstrate that although the effect of O2.- on Ca2+ efflux from RyR/Ca2+ release channel at higher concentrations ( >5 microM) is due to its ability to produce a loss in function of calmodulin thereby decreasing calmodulin inhibition, O2.- radicals at lower concentrations (<5 microM) may be able to stimulate Ca2+ release only in the presence of calmodulin from the SR via increased cADPR synthesis; it is also shown that cADPR is a modulator that can activate the Ca2+-release mechanism when it is in a sensitized state by the presence of calmodulin, possibly, at physiological concentration. In addition, the SR vesicles immediately upon addition of cADPR, but not NAD+, did exhibit Ca2+ efflux stimulation. When heart homogenate was incubated with O2.-, conversion of NAD+ into cADPR was stimulated; the reduction of homogenate Ca2+ uptake (by increasing Ca2+ efflux through RyR/Ca2+ release channel) occurred. Thus O2.- radical is responsible for cADPR formation from NAD+ in the cellular environment outside of the SR of heart muscle. The results presented here provide the first evidence of a messenger role for O2.- radical in cADPR-mediated Ca2+ mobilization in myocardium.

Intracellular effects of nitric oxide on force production and Ca2+ sensitivity of cardiac myofilaments.

The gaseous free radical nitric oxide (NO*) has been implicated in a wide range of physiological functions and also has a role in the pathogenesis of cellular injury. It has been suggested that NO* and its congeners may exert effects on actin-myosin crossbridge cycling by modulating critical thiols on the myosin head. To understand the mode and site of actin of NO* in myofibrils, the effects of the NO* donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl-1-propanamine (NOC-7) have been studied in Triton X-100-treated rabbit cardiac fibers, in which isometric force was measured at controlled degrees of activation. Experiments were undertaken after previous exposure of the preparations to NOC-7 (for 30 min). We found that NO* induced several alterations of myofibrillar function, i.e., decrease in Ca2+ sensitivity and Hill coefficient and potentiation of rigor contracture. We attributed the effect on rigor contracture to strong inhibition of myofibrillar creatine kinase (CK) activity, because it could be prevented by exogeneously added CK; such CK inactivation afforded by NO* may result in the myofibrillar ATP-to-ADP ratio. In further experiments, concentration of NO* released from NOC-7 was determined by the electron spin resonance spin-trapping technique; N-(dithiocarboxy)sarcosine-Fe2+ complex was used as the spin-trap. NO* at cumulative concentration of 0.69 microM was effective in producing both enhancement of rigor contracture and decrease of myofibrillar-bound CK activity; however, Ca2+-sensitivity (pCa50) was significantly decreased at >5.6 microM of NO*, suggesting a result from different mechanisms. Thus, the observed decrease in Ca2+ sensitivity seems to be associated with direct modification of the regulatory proteins by a relatively higher concentration of NO*, and possibly not via inhibition of myofibrillar CK activity. The data reported here indicate that CK may be a pathophysiologically main target for increased NO* formation at low molecular range in the disease state in cardiac muscle.

Differential sensitivity to hydroxyl radicals of pre- and postjunctional neurovascular transmission in the isolated canine mesenteric vein.

In some pathophysiological conditions, the first target of reactive oxygen intermediates is the vascular system. Superoxide anions, when generated in the vascular circulation, may then escape into the extracellular space via an anion channel and, following dismutation to hydrogen peroxide (H(2)O(2)), form hydroxyl radicals (HO(*)). In an attempt to understand the role of HO(*) in the regulation of transmission at the sympathetic neurovascular junction, the effect of HO(*) at nerve terminals was examined by measuring the amount of noradrenaline (NA) released from isolated, spirally cut, superfused canine mesenteric vein during basal and electrical stimulation (ES; 5Hz, 2ms, 9V); tension development evoked by ES was also recorded simultaneously. HO(*) was generated from Fenton's reagent (1. 5x10(-4)M H(2)O(2) plus 10(-4)M FeSO(4)); generation of HO(*) from H(2)O(2)/FeSO(4) in the superfusate was monitored by electron spin resonance spectroscopy using the spin-trap 5, 5-dimethyl-1-pyrroline-N-oxide throughout the experimental time course. Exposure to HO(*) of the helical strips produced an irreversible decrease in tension development evoked by ES with no effect on NA release, suggesting that the observed effect is elicited postjunctionally. The susceptibility of the processes of NA-mediated contraction to HO(*) may differ greatly from that of the NA release mechanism at the prejunctional site. Exposure of the strip preparation to HO(*) leads to a substantial stimulation of basal release of NA without affecting ES-evoked NA release, possibly due to enhanced non-exocytotic Ca(2+)-independent release elicited by HO(*). A direct demonstration of this concept was obtained by showing a significant increase in the basal response of NA release in Ca(2+)-free solution. The major conclusion of the present study is that HO(*) can damage NA-mediated contraction of the vascular preparations at the postjunctional site, and may selectively induce a non-exocytotic release of NA from the prejunctional site of sympathetic neurotransmission.

Endotoxin impairs the response of rabbit mesenteric artery to electrical stimulation via a prejunctional mechanism.

1. The effect of E. coli lipopolysaccharide (LPS) on sympathetic neuro-effector transmission was studied in the rabbit mesenteric artery. The experiments were performed on artery rings isolated 5 or 20 h after intravenous treatment with LPS or saline as well as on artery rings isolated from non-treated rabbits (for assessment of the effect of in vitro preincubation with LPS). In most experiments, neural elements in the arteries were stimulated electrically (10 V, 2 ms, 1-32 Hz). 2. Preincubation with LPS (10 micrograms ml-1) for 5 or 20 h had no effect on the contraction responses of endothelium-intact artery rings to electrical stimulation. In contrast, in vivo intravenous pretreatment with LPS (10 micrograms) led to an inhibition of the contraction; LPS elicited this effect when injected 20 h, but not 5 h, before the experiment. The effect of LPS was eliminated in artery rings isolated from animals receiving an inhibitor of protein synthesis (actinomycin D or cycloheximide) before treatment with LPS. LPS (injected 20 h before the experiment) had no effect on the concentration-response curves for exogenous noradrenaline and tyramine in endothelium-intact artery rings. 3. The inhibition of electrically induced contractions produced by LPS treatment in endothelium-intact artery rings was attenuated by atropine and yohimbine, but not by phentolamine. Yohimbine plus atropine restored the depressed contraction to the normal level. Clonidine and acetylcholine mimicked the effect of LPS in endothelium-intact artery rings isolated from saline-treated animals. 4. When steady-state contractions were induced by 5 min of stimulation at 16 Hz, acetylcholine or clonidine reduced the contraction in endothelium-denuded artery rings from both saline-treated rabbits and animals receiving LPS 20 h before the experiment. The reduction produced by acetylcholine or clonidine of the contraction in artery rings from LPS-treated rabbits was significantly greater than in artery rings from saline-treated animals.5. These results suggest that treatment of rabbits with LPS inhibits noradrenaline release from sympathetic nerve endings via increased sensitivity of both prejunctional inhibitory muscarinic receptors and x2-adrenoceptors in mesenteric arteries. They also suggest that the effect of LPS is independent of endothelial cells but linked to protein synthesis.

Inhibition by singlet molecular oxygen of the vascular reactivity in rabbit mesenteric artery.

1. The effects of reactive oxygen intermediates derived from photoactivated rose bengal on the vascular reactivity have been evaluated in rabbit mesenteric artery ring preparations. The artery rings were exposed to xanthene dye rose bengal (50 nM) illuminated (6,000 lux) at 560 nm for 30 min. Spin trapping studies with 2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with electron spin resonance spectrometry were also conducted in solution (and not within tissues) to determine quantitatively the reactive oxygen species generated from photoactivated rose bengal. 2. Contraction of the ring preparations induced by noradrenaline (10(-8) to 10(-4) M) was attenuated by previous exposure to photolysed rose bengal; the observation that the pD2 decreased without a significant reduction in maximum tension generation is consistent with the view that receptor dysfunction may be involved in the effect of photolysed rose bengal. 3. Prior exposure to photolysed rose bengal of the ring preparations inhibited the endothelium-dependent relaxation evoked by acetylcholine (10(-6) M) and calcium ionophore A23187 (10(-7) M), but not the endothelium-independent relaxation evoked by nitroglycerin (10(-6) M). 4. A variety of scavengers, superoxide dismutase (33 units ml-1), catalase (32 units ml-1) and 1,3-dimethyl-2-thiourea (DMTU, 10 mM), which should eliminate the superoxide anion radical, H2O2 and the hydroxyl radical, had no effect on the attenuated responses to noradrenaline and acetylcholine induced by photolysed rose bengal. In contrast, the inhibition of the observed effect of photolysed rose bengal was obtained with addition of histidine (25 mM), a singlet molecular oxygen quencher. 5. It was found that photolysis of rose bengal from a 1:2:2:1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct, which was effectively blunted by DMTU, superoxide dismutase and catalase whereas histidine was ineffective. The results of the electron spin resonance study also showed that a singlet molecular oxygen was produced by photoactivation of rose bengal; this was detected as singlet oxygen-TEMP product (TEMPO; 2,2,6,6-tetramethylpiperidine-N-oxyl). The formation of the TEMPO signal was strongly inhibited by histidine, but not by DMTU, superoxide dismutase and catalase. 6. It is suggested that the superoxide anion radical, H2O2 and hydroxyl radical are formed in addition to singlet molecular oxygen, and the data obtained from the present study indicate that singlet molecular oxygen is one of the most destructive oxygen species. Endothelium-dependent relaxation is quite vulnerable to singlet molecular oxygen. Singlet oxygen also depresses noradrenaline-induced contraction possibly via alpha-adrenoceptor dysfunction. This, in turn, may lead to vascular incompetence.

Inhibition by free radical scavengers and by cyclooxygenase inhibitors of the effect of acidosis on calcium transport by masseter muscle sarcoplasmic reticulum.

In vitro, arachidonic acid depressed calcium transport by sarcoplasmic reticulum (SR) in the homogenate of canine masseter muscle. This effect was inhibited by superoxide dismutase (SOD), a scavenger of the superoxide anion radial ( . O-2), at pH 7.0, and by SOD plus d-mannitol, a scavenger of hydroxyl free radical ( . OH), at pH 5.5. Indomethacin and 2-aminomethyl-4-tert-butyl-6-propionyl phenol (ONO-3144), a compound known to accelerate the conversion of prostaglandin G2 (PGG2) to PGH2 and scavenge free radicals, inhibited the effect of arachidonic acid at both pH 7.0 and pH 5.5. PGG2, but not PGH2, duplicated the effect of arachidonic acid. The effect of PGG2 on SR function was similar to that of exogenous free radicals generated from the xanthine-xanthine oxidase system. Incubation at pH 5.5, in the absence of an exogenous free-radical generating system, depressed SR calcium transport in the homogenate and in isolated SR. This effect in the homogenate was inhibited by indomethacin or by ONO-3144. At 10-min incubation at pH 5.5, SOD partially and temporarily reversed the depressant effect of acidosis. The addition of SOD plus d-mannitol completely reversed the system. d-Mannitol alone was ineffective. Arachidonic acid was able to mimic these effects of acidosis, except that arachidonic acid further depressed isolated SR calcium transport. These results demonstrate that acidosis can depress SR calcium transport in the homogenate of masseter muscle by an oxygen-free radical mechanism by the generation of . O-2 and . OH. Our results also demonstrate that significant oxygen radical generation can occur through the cyclooxygenase pathway of arachidonic acid metabolism at an acidotic pH in the cellular environment outside of the SR of the muscle cell, and seems to be responsible for the generation of the . OH derived from . O-2.

Susceptibility of caffeine- and Ins(1,4,5)P3-induced contractions to oxidants in permeabilized vascular smooth muscle.

Two principal pathways of Ca2+ release from the sarcoplasmic reticulum of excitable and non-excitable cells have been described: one pathway dependent on the second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and a second pathway sensitive to Ca2+ and regulated by caffeine and ryanodine. It was found that the Ca(2+)-pump activity of vascular smooth muscle sarcoplasmic reticulum is inhibited by superoxide anion radicals (O2.-); however, the effects of reactive oxygen intermediates on sarcoplasmic reticulum Ca2+ release in vascular muscle cells are not well defined. The purpose of the present study was to evaluate the effects of reactive oxygen intermediates generated from the hypoxanthine/xanthine oxidase reaction system on contractions induced by caffeine, Ins(1,4,5)P3 and norepinephrine in staphylococcal alpha-toxin-permeabilized rabbit mesenteric arteries. This system generates O2.-, H2O2, and hydroxyl radicals. We wished to identify which class of reactive oxygen intermediates is responsible for the associated loss of vascular smooth muscle contractile function. Caffeine and Ins(1,4,5)P3 produced a transient contraction when the sarcoplasmic reticulum of the permeabilized, preparations was preloaded with pCa 7.0 solution for 5 min before washing with 0.5 mM EGTA solution; norepinephrine also produced a transient contraction. Exposure of the preparations to hypoxanthine/xanthine oxidase (for 30 min) attenuated caffeine-induced contraction, but was without effect on Ins(1,4,5)P3-induced contraction. The observed effect of hypoxanthine/xanthine oxidase exposure was superoxide dismutase-inhibitable, suggesting O2.- involvement. Hypoxanthine/xanthine oxidase also inhibited norepinephrine-induced contraction. The effect of hypoxanthine/xanthine oxidase on norepinephrine contraction was protected by catalase, but not by superoxide dismutase and dimethyl sulfoxide; exogenously added H2O2 mimicked the effect of hypoxanthine/xanthine oxidase exposure. H2O2, added exogenously, was without effect on Ins(1,4,5)P3-induced contraction. It is suggested that the pathway of Ca2+ release from the sarcoplasmic reticulum dependent on Ins(1,4,5)P3 is insensitive to O2.-. Instead, caffeine-induced Ca2+ release mechanisms may be susceptible to O2.- and H2O2, rather than O2.- and hydroxyl radicals, may be the active agent in the norepinephrine-induced contraction. Our results are also consistent with the view that the attenuation by H2O2 of the norepinephrine-induced contraction may be linked to the receptor-associated pathway of Ins(1,4,5)P3 formation, but not to degradation processes of Ins(1,4,5)P3.

The effect of hypochlorous acid and hydrogen peroxide on coronary flow and arrhythmogenesis in myocardial ischemia and reperfusion.

The purpose of this study was to investigate the effect of the oxidants hypochlorous acid (HOCl) and hydrogen peroxide (H2O2) on the vulnerability of the myocardium to reperfusion-induced arrhythmias following global ischemia. After a 15 min equilibration period with or without experimental intervention, isolated perfused rat hearts in the Langendorff mode were made globally ischemic for 5 min by cross-clamping the aortic line. No dysrhythmias were evoked upon reperfusion at the 5 min global ischemia time period. HOCl or H2O2 were added to the perfusate 5 min into the equilibration period with a total exposure of 10 min. Global ischemia was then induced for 5 min followed by 10 min of reperfusion. A dose-response curve for HOCl (50-200 microM) indicated the development of idioventricular rhythms, in a concentration-dependent way. Furthermore, coronary flow of the hearts exposed to 100 and 200 microM HOCl, at 5 min post-reperfusion, was decreased; methionine (10 microM to 1 mM), an accepted scavenger for HOCl, prevented the responses to 200 microM HOCl, in a concentration-dependent manner. All hearts exposed to 200 microM H2O2 developed ventricular dysrhythmias during the reperfusion period. Coronary flow increased after 5 min of exposure to 200 microM H2O2 and remained elevated during reperfusion. It is concluded that toxic oxygen derived products are capable of increasing the susceptibility of the myocardium to reperfusion induced arrhythmias, and that although the electrical responses to exposure to those two oxidants were similar, the effects on the vasculature were not the same.

Reactive oxygen species participation in experimentally induced arthritis of the temporomandibular joint in rats.

In the temporomandibular joint (TMJ), it has been hypothesized that mechanical stresses lead to the oxidative stress of articular tissues. It has also been postulated that cells pertinent to arthritis-including endothelial cells and synovial cells-when stimulated by mechanical stresses and/or pro-inflammatory cytokines, promote oxidative damage. To determine the involvement of reactive oxygen species (ROS) in the diseased joint, we studied the generation of ROS in synovial fluid (SF) from interleukin-1alpha (IL-1alpha)-induced TMJ arthritis by electron spin resonance (ESR) spectroscopy, using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The TMJ arthritis was experimentally induced in rats by the injection of human recombinant IL-1alpha into the TMJ; control rats were treated with normal saline solution. We found that the detected radicals in the collected SF were identified as a 1:2:2:1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct. The ESR signal intensity of the hydroxyl radical-DMPO spin adduct in the SF from IL-1-treated rats was significantly higher than that from the control rats (P < 0.01). The results of ESR study also showed that hydroxyl radical (HO*) was increased in a time-dependent fashion in the presence of superoxide anion radical (O2*-) scavenger superoxide dismutase (SOD); the formation of DMPO-HO* was strongly inhibited by the iron chelater deferoxamine. We could measure higher levels of free iron (Fe2- and Fe3-) in the SF from TMJ arthritis than in that from controls (P < 0.05). Analysis of the data obtained from the present study suggests that the HO* radical detected in SF from IL-1-induced TMJ arthritis is generated via a modified Haber-Weiss reaction (biological Fenton reaction) in which O2*- can subsequently result in the production of H2O2 through dismutation reaction by SOD. Thus, HO* may be generated from the reaction of resultant H2O2 with free iron ions. The results presented here provide the first evidence of involvement of ROS in IL-1-induced TMJ arthritis.

Direct pharmacological action of vasoactive substances on pulpal blood flow: an analysis and critique.

An adequate blood supply to the dental pulp is essential to the health of the tooth; therefore, there have been a number of efforts to study pulpal blood flow and the factors which influence it. However, blood flow to the dental pulp is relatively inaccessible and apparently quite low. Consequently, it is difficult to obtain accurate flow measurements, partly owing to methodological difficulties with the small size of the tissue and its enclosure within rigid walls. In this study, the effects of locally applied vasoactive substances and their specific antagonists on pulpal blood flow have been examined by laser Doppler flowmetry. It is the purpose of this article to examine, in-depth, the involvement of endogenous vasoactive substances in the regulatory mechanism of blood flow within the dental pulp and expand our knowledge of pulpal microcirculatory hemodynamics.

Endogenous vasoactive substances and oxygen-derived free radicals in pulpal haemodynamics.

An adequate blood supply to the dental pulp is essential to the health of the tooth. A recent concept is that repeated stimulation of sensitive teeth may induce pulpal changes; this could occur through induction of neurogenic inflammation and alteration of pulpal blood flow. One possibility is that production of oxygen-derived free radicals at sites of inflammation contributes to alterations in local blood flow. The first target of free radicals, generated in several pathological processes, is the vascular system (essentially the endothelium). Although the exact mechanism by which free radicals induce changes in vascular conductance is still uncertain, they may act directly on vascular smooth muscle or modify vascular tone by interacting with the production and/or biological activity of endogenous vasoactive mediators. Recent data indicate that the oxygen-derived, free radical-generating system can decrease pulpal blood flow in the dog via endothelial dysfunction when applied locally in deep dentinal cavities. In addition to the part played by oxygen-derived free radicals, the measurement of pulpal blood flow and the effects of endogenous vasoactive substances on flow are discussed.

Dielectric investigation of electrically oriented ferroelectric smectic mixture CS-1013.

From dielectric spectroscopic study, a first-order ferroelectric phase transition has been observed in ferroelectric smectic mixture CS-1013 having the phase sequence Cr-SmC*-SmA-N*-Iso. Frequency (100 Hz-10 MHz) and temperature-dependent dielectric measurements have been performed on an electrically aligned sample (thickness 15+/-1 microm) gold coated on glass plates. In the unidirectionally aligned sample, two dielectric relaxation modes (Goldstone mode and soft mode) have been clearly observed in the ferroelectric SmC* phase while only one relaxation mode (soft mode) is visualized in the paraelectric SmA phase. Low-frequency molecular relaxation was also observed in the smectic phases. The experimental results have also been analyzed at different temperatures and biasing voltages for an understanding of the dynamics of dielectric processes in the ferroelectric phase. Finally, we proposed the "pseudospin" model for understanding the ferroelectric-antiferroelectric transition in liquid crystals. We associate the tilt angle straight theta and the pitch of the helix, respectively, with biaxial (b) and uniaxial (u) anisotropy parameters as fluctuating parameters around their stability limit (corresponding to the crystalline values). Here, the director acts as the pseudospin variable. This gives rise to a transverse Ising type (or anisotropic Heisenberg model under the mean-field approximation). It is then shown that such a model with fluctuations of (b) and (u) would explain the ferroelectric and antiferroelectric phase transitions in such liquid crystals. Using Landau theory and the stability conditions, we have also shown, in brief, the feasibility of different types of phase transitions in the ferroelectric liquid crystal system.

A case of nephrotic syndrome associated with traumatic chronic osteomyelitis.

A 19-year-old male developed nephrotic syndrome during the course of chronic osteomyelitis complicating a traumatic suppurative arthritis of the knee. Renal biopsy revealed severe mesangial proliferative glomerulonephritis, and immunofluorescent microscopy demonstrated the presence of IgA. Nephrotic syndrome remitted during the treatment for chronic osteomyelitis, suggesting a close association of the two conditions.

[Significance of urinary fibrin/fibrinogen degradation product (FDP) D-dimer measured by highly sensitive ELISA method with a new monoclonal antibody (D-D E72) in various renal diseases].

In order to clarify the abnormalities of the coagulation and fibrinolysis system in patients with various renal diseases, we produced a new monoclonal antibody for FDP (fibrin/fibrinogen degradation product) D-dimer (D-D E72). We also established a new highly sensitive method of enzyme-linked immunosorbent assay (ELISA) for urinary FDP D-dimer using this monoclonal antibody. The urine from 110, patients with various renal diseases was investigated for the FDP D-dimer. The results are summarized as follows: 1) Urinary FDP D-dimer in normal subjects was 0.69 +/- 0.60 ng/ml. 2) The level of urinary FDP D-dimer in patients with primary nephrotic syndrome and in patients with chronic renal failure was significantly higher than that of normal subjects, whereas the urinary FDP D-dimer levels in patients with diabetes mellitus were higher than those of normal subjects. 3) In the CGN and NS groups there was a tendency for an increase in the level of urinary FDP D-dimer in more active forms of the disease. 4) A significant correlation between urinary FDP D-dimer and urinary protein in the CGN and NS groups was demonstrated. 5) In all of the renal diseases investigated in this study, the ratio of urinary FDP D-dimer to total FDP was less than 4%.

[A study on the mechanism of phleboid olfactory function].

Phleboid olfactory test has wide clinical application for olfactory disturbance. Many unclarified aspects still remain concerning the mechanism of smell after an intravenous infusion of Alinamin. It is believed that when Alinamin is infused intravenously and byproducts are discharged from the blood into the alveoli, odorous substances reach the nasal cavity through exhalation. In order to clarify the mechanism of smell in more detail, we conducted the following experiments: 1) Cases of laryngectomy were examined to determine if intravenous infusion of Alinamin affected the olfactory organ. 2) Saliva specimens in healthy subjects were collected before and after intravenous infusion of Alinamin and examined for the presence of a secreted odorous substance using a functional test. 3) Healthy subjects who were inhibited in their sense of smell were examined for the presence of garlic odor after intravenous infusion of Alinamin. The results were as follows: 1) 80% of subjects of laryngectomy without cotton in the nares and 20% of subjects of laryngectomy with cotton in the nares perceived the garlic odor. 2) Garlic odorous substance was not recognized in saliva specimens collected before and after intravenous infusion of Alinamin. 3) The healthy subjects inhibited in their sense of smell and intravenously infused with Alinamin perceived the odor at the same time as exhalation. The above results indicated that the mechanism of smell after intravenous infusion of Alinamin occurred via exhalation, but not through the other routes.