Mechanisms of human insulin resistance and thiazolidinedione-mediated insulin sensitization.

Cellular and tissue defects associated with insulin resistance are coincident with transcriptional abnormalities and are improved after insulin sensitization with thiazolidinedione (TZD) PPARgamma ligands. We characterized 72 human subjects by relating their clinical phenotypes with functional pathway alterations. We transcriptionally profiled 364 biopsies harvested before and after hyperinsulinemic-euglycemic clamp studies, at baseline and after 3-month TZD treatment. We have identified molecular and functional characteristics of insulin resistant subjects and distinctions between TZD treatment responder and nonresponder subjects. Insulin resistant subjects exhibited alterations in skeletal muscle (e.g., glycolytic flux and intramuscular adipocytes) and adipose tissue (e.g., mitochondrial metabolism and inflammation) that improved relative to TZD-induced insulin sensitization. Pre-TZD treatment expression of MLXIP in muscle and HLA-DRB1 in adipose tissue from insulin resistant subjects was linearly predictive of post-TZD insulin sensitization. We have uniquely characterized coordinated cellular and tissue functional pathways that are characteristic of insulin resistance, TZD-induced insulin sensitization, and potential TZD responsiveness.

Antioxidant properties of isotorachrysone isolated from Rhamnus nakaharai.

Isotorachrysone inhibited iron-induced lipid peroxidation with an IC50 value of 1.64 +/- 0.08 microM in rat brain homogenates, and was comparable in potency to butylated hydroxytoluene and was more potent than alpha-tocopherol or desferrioxamine. The mechanism of antioxidant properties were then examined. Isotorachrysone could scavenge the stable free radical diphenyl-p-picrylhydrazyl. And it was an efficient direct scavenger of water-soluble peroxyl radicals with stoichiometry factor of 0.53 +/- 0.05 in the aqueous phase and also toward lipid-soluble peroxyl radicals in tissue homogenates. The oxygen consumption during peroxidation induced by radicals on human erythrocyte ghosts was suppressed by isotorachrysone. Furthermore, it was reactive towards superoxide anion with a second-order rate constant of 5.06 +/- 0.65 x 10(5) M-1 S-1. But it did not react with hydrogen peroxide detected within the sensitivity limit of our assay. Using ascorbate/iron ion/H2O2 as a hydroxyl radical generating system and deoxyribose as a probe, isotorachrysone was effective with hydroxyl radicals with a second-order rate constant of 3.88 +/- 0.54 x 10(11) M-1 S-1 under stimulation by iron-EDTA. On the other hand, isotorachrysone retarded the peroxidation of human low density lipoprotein (LDL) initiated by both aqueous and lipophilic peroxyl radicals. And it also suppressed copper-catalyzed human LDL oxidation, as measured by fluorescence intensity, electrophoretic mobility, and thiobarbituric acid-reactive substances formation in a concentration-dependent manner. Our results show that isotorachrysone is potentially an effective and versatile antioxidant, and can help protecting LDL against oxidation.

Cinnamophilin as a novel antiperoxidative cytoprotectant and free radical scavenger.

The antioxidant properties of cinnamophilin were evaluated by studying its ability to react with relevant reactive oxygen species, and its protective effect on cultured cells and biomacromolecules under oxidative stress. Cinnamophilin concentration-dependently suppressed non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 8.0+/-0.7 microM and iron ion/ADP/ascorbate-initiated rat liver mitochondrial lipid peroxidation with an IC50 value of 17.7+/-0.2 microM. It also exerted an inhibitory activity on NADPH-dependent microsomal lipid peroxidation with an IC50 value of 3.4+/-0.1 microM without affecting microsomal electron transport of NADPH-cytochrome P-450 reductase. Both 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azo-bis(2-amidinopropane) dihydrochloride-derived peroxyl radical tests demonstrated that cinnamophilin possessed marked free radical scavenging capacity. Cinnamophilin significantly protected cultured rat aortic smooth muscle cells (A7r5) against alloxan/iron ion/H2O2-induced damage resulting in cytoplasmic membranous disturbance and mitochondrial potential decay. By the way, cinnamophilin inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity and thiobarbituric acid-reactive substance formation in a concentration-dependent manner. On the other hand, it was reactive toward superoxide anions generated by the xanthine/xanthine oxidase system and the aortic segment from aged spontaneously hypertensive rat. Furthermore, cinnamophilin exerted a divergent effect on the respiratory burst of human neutrophil by different stimulators. Our results show that cinnamophilin acts as a novel antioxidant and cytoprotectant against oxidative damage.

Protection of oxidative hemolysis by demethyldiisoeugenol in normal and beta-thalassemic red blood cells.

The purpose of this study was to evaluate the ability of demethyldiisoeugenol to protect normal and beta-thalassemic human red blood cells (RBCs) against oxidative damage in vitro. Oxidative hemolysis and lipid peroxidation of normal and beta-thalassemic human RBCs induced by aqueous peroxyl radical were suppressed by demethyldiisoeugenol in a concentration-dependent manner. The formation of proteins with high molecular weight and concomitant decrease of the low-molecular-weight proteins of RBCs challenge with aqueous peroxyl radical were inhibited by demethyldiisoeugenol. It also prevented the shortening of the Russell's viper venom (RVV)-clotting time mediated by prelytic radical-treated RBCs. In contrast, demethyldiisoeugenol inhibited oxidative hemolysis but not those metHb and ferrylHb formations caused by hydrogen peroxide (H2O2) in normal RBCs. Furthermore, demethyldiisoeugenol did not prevent the consumption of the cytosolic antioxidant, glutathione (GSH), in radical-treated RBCs. It also did not cause of a loss of sulfhydryl group during incubation with GSH. However, the diphenyl-2-picrylhydrazyl (DPPH) scavenging activity of demethyldiisoeugenol was dramatically increased in the presence of GSH. These results imply that demethyldiisoeugenol can regenerate from its oxidized form to its active reduced form in the presence of GSH. It may be useful in diminishing oxidative damage to pathological RBCs.

Mechanisms involved in the antiplatelet activity of Staphylococcus aureus lipoteichoic acid in human platelets.

In this study, gram-positive Staphylococcus aureus lipoteichoic acid (LTA) dose-dependently (0.1-1.0 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LTA also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. LTA (0.5 and 1.0 microg/ml) also significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, LTA (0.1-1.0 microg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. Rapid phosphorylation of a platelet protein of Mr. 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (30 nM). This phosphorylation was markedly inhibited by LTA (0.5 and 1.0 microg/ml) within a 10-min incubation period. These results indicate that the antiplatelet activity of LTA may be involved in the following pathways: LTA's effects may initially be due to induction of conformational changes in the platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in gram-positive septicemic and endotoxemic patients.

Inhibition of collagen-induced platelet aggregation and adhesion by a pseudocyanide derivative of avicine isolated from Zanthoxylum integrifoliolum Merr.

Avicine pseudocyanide, a derivative of avicine isolated from Zanthoxylum integrifoliolum Merr., inhibited collagen-induced platelet aggregation and release reaction in a concentration-dependent manner. Trimucytin is a collagen-like snake venom protein isolated from Trimeresurus mucrosquamatus. Avicine pseudocyanide also inhibited trimucytin (1 microgram/mL)-induced platelet aggregation and release reaction concentration dependently. The IC50 values of avicine pseudocyanide on collagen (10 micrograms/mL)- and trimucytin (1 microgram/mL)-induced platelet aggregation were 47.3 +/- 4.1 and 62.5 +/- 5.6 microM, respectively. Avicine pseudocyanide at a concentration of 300 microM inhibited less than 30% of platelet aggregation induced by ADP (20 microM), AA (100 microM), U46619 (1 microM), PAF (2 ng/mL) and thrombin (0.1 U/mL). The concentration-response curve of collagen-induced platelet aggregation was shifted to the right by avicine pseudocyanide (20-100 microM) concentration dependently. The Schild plot showed that pA2 and pA10 values of avicine pseudocyanide were 4.8 and 4.3, respectively, with slope of -1.9. Avicine pseudocyanide also inhibited collagen (10 micrograms/mL)-induced aggregation of rabbit whole blood with an IC50 of 145 +/- 13 microM. Collagen-induced thromboxane B2 formation was also inhibited by avicine pseudocyanide in a concentration-dependent manner with a maximal effect at 100 microM. However, arachidonic acid (AA)-induced thromboxane B2 and prostaglandin D2 formations were only partially suppressed by a high concentration of avicine pseudocyanide (300 microM). Avicine pseudocyanide (100 microM) inhibited the [3H]inositol monophosphate formation and the rise of intracellular Ca2+ concentration caused by collagen but not those caused by AA, U46619, platelet-activating factor and thrombin. In the presence of prostaglandin E1, Mg(2+)-dependent platelet adhesion to collagen was inhibited by avicine pseudocyanide with an IC50 of 278 +/- 16 microM. These data indicate that avicine pseudocyanide is an inhibitor of collagen-induced platelet aggregation and platelet-collagen adhesion.

Antithrombotic effects of tetramethylpyrazine in in vivo experiments.

In this study, tetramethylpyrazine (TMPZ) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 40 and 80 microg/g. In addition, intravenous injection of TMPZ (10 microg/g) significantly prolonged the bleeding time by approximately 1.5-fold compared with normal saline in severed mesenteric arteries of rats. Continuous infusion of TMPZ (1 microg/g per min) for 10 minutes also significantly increased the bleeding time approximately 1.6-fold, and the bleeding time returned to baseline within 60 minutes after cessation of TMPZ infusion. On the other hand, platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pre-treated intravenously with fluorescein sodium (10 microg/kg). When it was intravenously injected, TMPZ (250 microg/g) significantly prolonged the latent period of the induction of platelet plug formation in mesenteric venules. TMPZ (250 microg/g) prolonged occlusion time approximately 1.4-fold (183 +/- 18 seconds) compared with that of normal saline (132 +/- 14 seconds). Furthermore, aspirin (300 microg/g) showed similar activity in the prolongation of occlusion time in this experiment. In conclusion, these results suggest that TMPZ has effective antithrombotic activity in vivo and may be a potential therapeutic agent for arterial thrombosis but must be assessed further for toxicity.

N-allylsecoboldine as a novel antioxidant against peroxidative damage.

N-Allylsecoboldine was evaluated for antioxidant properties by studying its ability to react with relevant reactive oxygen species, and its protective effect on human erythrocytes under oxidative stress. Using brain homogenates, we found that N-allylsecoboldine dose dependently inhibited lipid peroxidation (IC50 = 4.80 +/- 0.16 microM) and markedly scavenged stable nitrogen-centered radicals. N-Allylsecoboldine was a very efficient scavenger for inhibiting peroxyl radical-mediated destruction of B-phycoerythrin, with a stoichiometry factor of 4.40 +/- 0.59. It also trapped the hydroxyl radicals with a second-order rate constant of 6.92 +/- 0.86 x 10(9) M-1 S-1. Additionally, human erythrocyte oxidative hemolysis induced by aqueous peroxyl radical or hydrogen peroxide was suppressed by N-allylsecoboldine. It not only attenuated the extent of lipid peroxidation but also decreased the formation of the high-molecular weight proteins and degradation of the band 6 protein in radical-treated erythrocytes. It also inhibited the shortening of Russell's viper venom-clotting time mediated by prelytic radical-treated erythrocytes. In the presence of exogenous oxidative stress, hemolysis and lipid peroxidation were significantly enhanced in beta-thalassemic erythrocytes, as compared to the normal control. These elevated detrimental effects could be prevented by N-allylsecoboldine. It is concluded that N-allylsecoboldine may act as an effective antioxidant and protect cells against oxidative damage.

Magnolol reduces myocardial ischemia/reperfusion injury via neutrophil inhibition in rats.

The accumulation of oxygen-free radicals and activation of neutrophils are strongly implicated as important pathophysiological mechanisms mediating myocardial ischemia/reperfusion injury. It has been proven that various antioxidants have cardioprotective effects. Magnolol, an active component extracted from the Chinese medicinal herb Magnolia officinalis, possesses potent antioxidant and free radical scavenging activities. In this study, the cardioprotective activity of magnolol was evaluated in an open-chest anesthetized rat model of myocardial ischemia/reperfusion injury. The results demonstrated that pretreatment with magnolol (0.2 and 0.5 microg/kg, i.v. bolus) at 10 min before 45 min of left coronary artery occlusion, significantly suppressed the incidence of ventricular fibrillation and mortality when compared with the control group. Magnolol (0.2 and 0.5 microg/kg) also significantly reduced the total duration of ventricular tachycardia and ventricular fibrillation. After 1 h of reperfusion, pretreatment with magnolol (0.2 and 0.5 microg/kg) caused a significant reduction in infarct size. In addition, magnolol (0.2 microg/kg) significantly reduced superoxide anion production and myeloperoxidase activity, an index of neutrophil infiltration in the ischemic myocardium. In addition, pretreatment with magnolol (0.2 and 0.5 microg/kg) suppressed ventricular arrhythmias elicited by reperfusion following 5 min of ischemia. In vitro studies of magnolol (5, 20 and 50 microM) significantly suppressed N-formylmethionyl-leucyl-phenylalanine (fMLP; 25 nM)-activated human neutrophil migration in a concentration-dependent manner. It is concluded that magnolol suppresses ischemia- and reperfusion-induced ventricular arrhythmias and reduces the size of the infarct resulting from ischemia/reperfusion injury. This pronounced cardioprotective activity of magnolol may be mediated by its antioxidant activity and by its capacity for neutrophil inhibition in myocardial ischemia/reperfusion.

Antiplatelet activity of Staphylococcus aureus lipoteichoic acid is mediated through a cyclic AMP pathway.

In this study, Gram-positive Staphylococcus aureus lipoteichoic acid (LTA) dose dependently (0.1-1.0 microg/mL) and time dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., thrombin and collagen). LTA also dose dependently inhibited intracellular Ca(2+) mobilization in human platelets stimulated by collagen. In addition, LTA (0.5 and 1.0 microg/mL) dose dependently increased the formation of cyclic AMP but not cyclic GMP in platelets. LTA (0.5 and 1.0 microg/mL) did not significantly increase the production of nitrate within a 10-min incubation period. Rapid phosphorylation of a platelet protein of M(r) 47,000, a marker of protein kinase C activation, was triggered by PDBu (0.03 microM). This phosphorylation was dose dependently inhibited by LTA (0.5 and 1.0 microg/mL) within a 10-min incubation period. Furthermore, LTA (0.5 and 1.0 microg/mL) also inhibited platelet aggregation induced by PDBu (0.03 microM) in human platelets. These results indicate that the antiplatelet activity of LTA may be involved in the increase of cyclic AMP, leading to inhibition of intracellular Ca(2+) mobilization and protein kinase C activity. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients.

Mechanisms involved in the inhibition of neointimal hyperplasia by abciximab in a rat model of balloon angioplasty.

Monoclonal antibodies raised against beta(3) integrin are able to inhibit the binding of ligands to certain beta(3) integrins such as alpha(IIb)beta(3) (glycoprotein IIb/IIIa complex) and alpha(v)beta(3) (vitronectin receptor) and as such are inhibitors of platelet aggregation and smooth muscle cell (SMC) migration, both of which are involved in neointimal hyperplasia. The present study was designed to explore the detailed mechanisms of abciximab (Reopro), a monoclonal antibody (mAb) raised against alpha(IIb)beta(3) integrin in neointimal hyperplasia. In this study, carotid arteries of Wistar rats were damaged, and neointimal hyperplasia and lumen occlusion was determined at different time points. Abciximab was administered intravenously by an implanted osmotic pump. Abciximab (0.25 mg/kg/day) time-dependently inhibited both neointimal hyperplasia and lumen occlusion after angioplasty in carotid arteries of rats. Furthermore, the electromicrographs highlighted that SMCs were phenotypically different from the typical contractile, spindle-shaped SMCs normally seen in uninjured vessel walls. Platelet-derived growth factor (PDGF)-BB was strongly produced in thrombus formation and neointimal SMCs after angioplasty, while abciximab significantly reduced PDGF-BB expression in vessel lumens and neointimal SMCs after angioplasty. Balloon angioplasty caused a significant increase of nitrate and cyclic GMP as compared with sham-operated rats. Infusion of abciximab (0.25 mg/kg/day) did not significantly change. Furthermore, the plasma level of thromboxane B(2) (TxB(2)) obviously increased after angioplasty, while abciximab markedly suppressed the elevation of plasma TxB(2) concentration. The results indicate that abciximab effectively prevents neointimal hyperplasia, possibly through the following 2 mechanisms: (1) Abciximab binds to alpha(IIb)beta(3) integrin on platelet membranes resulting in inhibition of platelet adhesion, secretion, and aggregation in injured arteries, followed by inhibition of thromboxane A(2) formation and PDGF-BB release from platelets. (2) Abciximab may also bind to alpha(v)beta(3) integrin on SMCs, thus, subsequently inhibiting cell migration and proliferation.

Comparison of the relative activities of alpha-tocopherol and PMC on platelet aggregation and antioxidative activity.

In this study, PMC (2,2,5,7,8-pentamethyl-6-hydroxychromane), a potent antioxidant derived from alpha-tocopherol, dose-dependently inhibited agonist-induced platelet aggregation in human platelet-rich plasma. PMC is over 5-10 times more potent than alpha-tocopherol in inhibiting human platelet aggregation. Moreover, PMC (25-350 microM) dose-dependently reduced the relative fluorescence intensity of platelet membrane tagged with diphenylhexatriene (DPH). PMC is about 6-times more potent than alpha-tocopherol on this effect. Furthermore, antioxidative activity of PMC was investigated using two in vitro models. PMC inhibited non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 0.21+/-0.05 microM. It was more potent than alpha-tocopherol or other classical antioxidants. PMC also scavenged the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The concentration of PMC resulting in a decrease of 0.20 in the absorbance of DPPH was about 12.1+/-3.6 microM, was comparable in potency to alpha-tocopherol, butylated hydroxytoluence and Trolox. The antiplatelet activity of PMC may possibly be due initially to an increase in fluidity of the platelet membrane followed by inhibition of platelet aggregation. Our results indicate that PMC is a potentially effective antioxidant and antiaggregating agent, and could be helpful the design of compounds with more clinical effectiveness.

The Antimicrobial Effect of Silver Ion Impregnation into Endodontic Sealer against Streptococcus mutans.

Pulpal and periradicular diseases are primarily caused by bacterial invasion of the root canal system as a result of caries progression. The presence of residual bacteria at the time of root canal completion (obturation) is associated with significantly higher rate of treatment failure. Re-infection of obturated root canals can be potentially prevented by enhancing the antibacterial activities of root canal obturation materials. We evaluated, in an in vitro model, the antimicrobial efficacy of silver ions added to a common endodontic sealer. For that purpose we performed growth inhibition studies and bacterial viability tests. We measured the zone of inhibition, optical density and performed confocal laser scanning microscopy. Our results show that the silver ions enhance the antimicrobial activity of the root canal sealer against Streptococcus mutans. This study approach may hold promise for studying other biologically based therapies and therefore increasing the success rate of routine orthograde root canal treatment.

Acquired genital smooth-muscle hamartoma. A case report.

Smooth-muscle hamartoma is an uncommon, usually congenital malformation of smooth-muscle origin. When it is acquired, it may be confused with Becker's nevus with a prominent smooth-muscle component. Over the past 3 decades, only two cases of acquired smooth-muscle hamartoma without the concomitant appearance of hairy-pigmented lesions have been reported. We describe a third case. To the best of our knowledge, this is the first case of smooth-muscle hamartoma to occur in the scrotum.

Low-dose danazol in the treatment of livedoid vasculitis.

BACKGROUND: Livedoid vasculitis is characterized clinically by smooth or depressed ivory-white scars surrounded by hyperpigmentation and telangiectasia with or without preceding purpuric infiltrated papules and plaques and histologically by intravascular deposition of fibrin. Its etiology remains obscure and therapy very difficult. OBJECTIVE: Our purpose was to test the efficacy of low-dose danazol in the treatment of livedoid vasculitis. METHODS: Seven patients with active lesions of livedoid vasculitis were treated with low-dose danazol (200 mg, orally, daily). Laboratory coagulation and fibrinolysis parameters, including antithrombin III, protein C, protein S, tissue plasminogen activator, plasminogen, alpha 2-antiplasmin and fibrinogen, were evaluated before and during the therapy. RESULTS: Six of the 7 patients completed the treatment. After the therapy, all 6 patients had rapid cessation of new lesion formation, prompt reduction in their pain and healing of ulcers. A significant elevation of plasminogen and a decrease in fibrinogen levels were noted 1 month after initiation of the therapy (p = 0.028). The level of fibrinogen seemed to parallel the disease activity in individual patients. In addition, in most of these patients, the levels of antithrombin III, protein C, protein S and alpha 2-antiplasmin tended to increase after the treatment. However, the differences were not statistically significant. Abnormalities of tissue plasminogen activator levels were less consistent. Low-dose danazol was well tolerated without major side effects. CONCLUSION: We concluded that low-dose danazol was effective in the treatment of livedoid vasculitis, without unacceptable side effects.

Atypical mycobacterial cervical lymphadenitis associated with Sweet's syndrome.

We report the case of a 52-year-old woman with a non-tuberculous (atypical) mycobacterial cervical lymphadenitis, caused by Mycobacterium fortuitum, in association with Sweet's syndrome. The cervical lymphadenitis was resistant to medical treatment, and the Sweet's syndrome occurred intermittently. Systemic steroid treatment was required to control the cutaneous symptoms.

Plaque-type blue nevus on the face: a variant of Ota's nevus?

We describe a plaque-type blue nevus that had been present since birth on the right cheek of a 22-year-old man. It was characterized by several dark blue macules and papules with intervening areas of faint blue discoloration. Histologic examination showed a common blue nevus and a mongolian spot-like dermal melanocytosis in the dark blue and intervening faint blue pigmentary lesions, respectively.

Identification of a molecule of Porphyromonas gingivalis that binds to Streptococcus gordonii.

The molecules that mediate the adherence of Porphyromonas gingivalis, a periodontal pathogen, to Streptococcus gordonii, a commensal plaque organism, were investigated. Outer membrane proteins of P. gingivalis were labelled with biotin, extracted by EDTA and reacted with S. gordonii cells. Interactive porphyromonas components were identified by SDS-PAGE of the S. gordonii cells followed by electroblotting and visualization of the adsorbed porphyromonas molecules with streptavidin-alkaline phosphatase. A P. gingivalis molecule of 35 kDa bound to S. gordonii. Monospecific polyclonal antibodies to the 35 kDa protein inhibited binding of P. gingivalis to S. gordonii by 71%. The antibodies also reacted with the P. gingivalis fimbriae, indicating that the 35 kDa molecule is antigenically related to, or associated with, the fimbriae.