Double-stranded RNA induces the synthesis of retinoic acid-inducible gene-I in vascular endothelial cells.

Viral infection induces various responses in vascular endothelial cells. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), and treatment of cells with poly IC mimics the viral infection to the cells. Retinoic acid-inducible gene-I (RIG-I) is a protein belonging to the DExH-box family and designated as a putative RNA helicase. RIG-I is considered to play a role in antiviral responses through the regulation of gene expressions. In the present study, the authors treated human umbilical vein endothelial cells (HUVECs) with poly IC and found that poly IC induced the expression of RIG-I. The poly IC-induced RIG-I expression was inhibited by the preincubation of the cells with 2-aminopurine, an inhibitor of dsRNA-dependent protein kinase (PKR). Immunohistochemical examination revealed high levels of RIG-I immunoreactivity in vascular endothelial cells in the thalamus from rats inoculated with hantavirus. Induction of RIG-I by poly IC may be involved in the antiviral responses in endothelial cells.

Involvement of retinoic acid-inducible gene-I in the IFN-{gamma}/STAT1 signalling pathway in BEAS-2B cells.

Bronchial epithelial cells play an important role in airway host defence, and interferon (IFN)-gamma controls immune reactions by regulating the expression of various genes in bronchial epithelial cells. Signal transducer and activator of transcription 1 (STAT1) is the key transcriptional factor in IFN-gamma signalling. Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family of proteins and designated a putative RNA helicase. RNA helicases play diverse roles in regulation of gene expression and cellular functions, and RIG-I is implicated in antiviral responses. The aim of the present study was to investigate the effect of IFN-gamma on RIG-I expression in a cell line derived from human bronchial epithelial cells, BEAS-2B. Induction of RIG-I in response to IFN-gamma was found in BEAS-2B cells. Induction of RIG-I by IFN-gamma was also demonstrated in another pulmonary epithelial cell line, NCI-H292. Transfection of BEAS-2B cells with RIG-I complementary DNA resulted in the upregulation of STAT1. Induction of IFN-gamma-inducible protein 10 by IFN-gamma was enhanced in the cells overexpressing RIG-I. It is concluded that retinoic acid-inducible gene-I may play an important role in the regulation of immunological reactions in bronchial epithelial cells elicited by interferon-gamma.

Neurosyphilis showing transient global amnesia-like attacks and magnetic resonance imaging abnormalities mainly in the limbic system.

We report a case of neurosyphilis with transient global amnesia (TGA)-like attacks on the first presentation. MRI abnormalities in bilateral limbic systems, including a few lesions in the basal ganglia and thalamus, were identified. Depression and dementia became apparent, accompanied by a high treponemal antibody titer and mild cortical atrophy. Antisyphilitic therapy brought about mild improvement, and the MRI abnormalities decreased.

Hyperhomocysteinemia impairs angiogenesis in response to hindlimb ischemia.

Hyperhomocysteinemia (HH) is an independent risk factor for atherosclerosis, including peripheral arterial occlusive disease (PAOD). Because angiogenesis and collateral vessel formation are important self-salvage mechanisms for ischemic PAOD, we examined whether HH modulates angiogenesis in vivo in a rat model of hindlimb ischemia. Rats were divided into 3 groups: the control group was given tap water, the HH group was given water containing L-methionine (1 g x kg(-1) x d(-1)), and the HH+L-arg group was given water containing methionine (1 g x kg(-1) x d(-1)) and l-arginine (2.25 vol%). At day 14 of the dietary modifications, the left femoral artery and vein were excised, and the extent of angiogenesis and collateral vessels in the ischemic limb were examined for 4 weeks. Plasma homocysteine levels significantly increased (P:<0.001), and plasma and tissue contents of nitrite+nitrate as well as tissue cGMP levels significantly decreased in the HH group compared with the control group (P:<0.01). Laser Doppler blood flowmetry (LDBF) revealed a significant decrease in the ischemic/normal limb LDBF ratio in the HH group at days 7, 14, 21, and 28 (P:<0.01 versus control). Angiography revealed a significant decrease in the angiographic score in the HH group at day 14 (P:<0.001 versus control). Immunohistochemistry of ischemic tissue sections showed a significant reduction in the capillary density in the HH group (P:<0. 001 versus control). Oral l-arginine supplementation in rats with HH (HH+L-arg) restored the decreased plasma and tissue nitrite+nitrate and cGMP contents (P:<0.05) as well as angiogenesis, as assessed by LDBF (P:<0.05 versus HH), angiographic score (P:<0.01 versus HH), and capillary density (P:<0.001 versus HH). In summary, HH impaired ischemia-induced angiogenesis and collateral vessel formation in a rat model of hindlimb ischemia in vivo. The mechanism of the HH-induced impairment of angiogenesis might be mediated in part by a reduced bioactivity of endogenous NO in the HH state.

Hypercholesterolemia inhibits angiogenesis in response to hindlimb ischemia: nitric oxide-dependent mechanism.

BACKGROUND: Endothelium-derived nitric oxide (EDNO) plays an important role in the regulation of angiogenesis, whereas hypercholesterolemia (HC) impairs EDNO release. We examined the hypothesis that HC may inhibit ischemia-induced angiogenesis by inhibition of EDNO in a rat model of unilateral hindlimb ischemia and that oral L-arginine supplementation, a substrate for NO synthase, may prevent HC-related impairment of angiogenesis. METHODS AND RESULTS: Male Sprague-Dawley rats were fed (A) standard diet (control), (B) 2% high-cholesterol diet (HC group), or (C) high-cholesterol diet with oral L-arginine (2.25% in drinking water) (HC+L-arg group). At 2 weeks of the dietary intervention, unilateral limb ischemia was surgically induced in all animals. Dietary HC groups (B and C) revealed elevated total and LDL cholesterol levels compared with control animals. Laser Doppler blood flow analyses showed significant decreases in the ischemic/normal limb blood flow ratio in the HC group compared with controls (P:<0.05) when followed up until 4 weeks after surgery. Selective angiography and immunohistochemical analyses in the ischemic limb at postoperative day 14 revealed significantly lower angiographic scores (P:<0.01) and capillary densities (P:<0.01) in the HC group than controls, which were associated with decreased tissue contents of NO(x) and cGMP. Oral L-arginine supplementation (HC+L-arg) significantly improved all parameters of the laser Doppler blood perfusion ratio, angiographic scores, and capillary densities (P:<0.01 versus HC group), which were accompanied by significant elevations in serum L-arginine levels and tissue NO(x) and cGMP contents. CONCLUSIONS: Collateral vessel formation and angiogenesis in response to hindlimb ischemia were significantly attenuated in rats with dietary HC. The mechanism may be related to the reduced NO bioactivity in the ischemic tissues. Augmentation of the tissue NO activity by oral L-arginine supplementation restored the impaired angiogenesis in HC.

Tetrahydrobiopterin restores endothelial function in long-term smokers.

OBJECTIVES: We sought to test whether tetrahydrobiopterin (BH4) supplementation improves nitric oxide (NO) bioactivity in smokers. BACKGROUND: In smokers, endothelium-derived NO bioactivity is impaired. BH4 is an essential cofactor of NO synthase, and its deficiency decreases NO bioactivity. METHODS: Sapropterin hydrochloride, an active analogue of BH4 (2 mg/kg body weight), was administered orally to healthy male smokers and age-matched nonsmokers. Before and 3 and 24 h after sapropterin, we measured plasma levels of BH4 and examined flow-mediated vasodilation (FMV) of the brachial artery by high resolution ultrasonography, a noninvasive test of endothelial function. RESULTS: Basal plasma levels of BH4 were not different between smokers and nonsmokers. Sapropterin administration increased plasma levels of BH4 by threefold at 3 h, which returned to the baseline at 24 h. Before sapropterin, FMV was significantly smaller in smokers (p = 0.0002). Sapropterin significantly augmented endothelium-dependent vasodilation in smokers, but did not affect it in nonsmokers (p = 0.001 by analysis of variance [ANOVA]). Coadministration of N(G)-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor (20 micromol), into the brachial artery completely abolished the vasodilatory effects of sapropterin (p = 0.002 by ANOVA). Endothelium-independent vasodilation by glyceryl trinitrate was not different between smokers and nonsmokers and was not altered by BH4. CONCLUSIONS: We demonstrated that BH4 supplementation improved bioactivity of endothelium-derived NO in smokers. These observations strongly suggest that decreased NO-dependent vasodilation in smokers could be related to reduced bioactivity of BH4.

Endothelial dysfunction by acute hyperhomocyst(e)inaemia: restoration by folic acid.

Recent evidence demonstrates that hyperhomocyst(e)inaemia is a novel risk factor for cardiovascular diseases. In patients with chronic hyperhomocyst(e)inaemia, endothelial function is impaired. However, whether hyperhomocyst(e)inaemia per se is a cause or an epiphenomenon of endothelial dysfunction remains unknown. In this study, we examined the effects of methionine-induced acute hyperhomocyst(e)inaemia on human endothelial function. In healthy volunteers we administered methionine (0.1 g/kg body weight, per os), a substrate of homocyst(e)ine, with or without folic acid (20 mg, per os) and examined flow-mediated vasodilatation of the brachial artery by high-resolution ultrasonography as a non-invasive measure of endothelial function. We also measured plasma levels of homocyst(e)ine before and 3, 8 and 24 h after methionine loading. Methionine administration increased plasma levels of homocyst(e)ine by four times the basal level at 8 h (P<0.0001, ANOVA). The plasma levels returned to baseline at 24 h. Flow-mediated vasodilatation was significantly decreased to half of the baseline value at 8 h and returned to baseline at 24 h (P<0.0001, ANOVA), whereas endothelium-independent vasodilatation by glyceryl trinitrate was not affected by the methionine loading. Co-administration of folic acid did not attenuate methionine-induced hyperhomocyst(e)inaemia but completely prevented endothelial dysfunction. Our results suggest that in humans a methionine-rich diet may acutely impair endothelial function, which can be prevented by folic acid supplementation.

Chronic inhibition of nitric oxide in central nervous system does not cause hypertension.

Acute inhibition of nitric oxide (NO) synthase in the brain causes elevation of blood pressure and sympathetic excitation under anesthetized conditions. To investigate chronic effects of NO synthase inhibition in the central nervous system on blood pressure regulation in conscious unrestrained animals, we administered NG-monomethyl-L-arginine (L-NMMA), a potent NO synthase inhibitor, at low (22.5 mumol/kg) and high (67.5 mumol/kg) doses for 1 wk into the cisterna magna with an osmotic pump and measured mean arterial pressure (MAP) and heart rate (HR) by a telemetry method. The same dose of NG-monomethyl-D-arginine (D-NMMA), an inactive isomer of L-NMMA, was administered to control rats. Chronic intracisternal administration of low-dose L-NMMA significantly decreased the brain nitrite/nitrate and NO metabolite contents as compared with D-NMMA (p < 0.05). However, MAP and its variability, HR and its variability, and plasma norepinephrine levels did not differ between the two groups of rats at either low- or high-dose treatment. Thus, chronic NO synthase inhibition in the central nervous system did not affect systemic hemodynamics or plasma norepinephrine concentrations despite the inhibition of brain NO. Our results suggest that endogenous NO in the central nervous system, at least as a whole, may not affect the systemic hemodynamics of chronic unanesthetized rats.

Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), the most common cause of sudden death in the young, is an autosomal dominant disease characterized by ventricular hypertrophy accompanied by myofibrillar disarrays. Linkage studies and candidate-gene approaches have demonstrated that about half of the patients have mutations in one of six disease genes: cardiac beta-myosin heavy chain (c beta MHC), cardiac troponin T (cTnT), alpha-tropomyosin (alpha TM), cardiac myosin binding protein C (cMBPC), ventricular myosin essential light chain (vMLC1) and ventricular myosin regulatory light chain (vMLC2) genes. Other disease genes remain unknown. Because all the known disease genes encode major contractile elements in cardiac muscle, we have systematically characterized the cardiac sarcomere genes, including cardiac troponin I (cTnI), cardiac actin (cACT) and cardiac troponin C (cTnC) in 184 unrelated patients with HCM and found mutations in the cTnI gene in several patients. Family studies showed that an Arg145Gly mutation was linked to HCM and a Lys206Gln mutation had occurred de novo, thus strongly suggesting that cTnI is the seventh HCM gene.

Antinociceptive components of Ganoderma lucidum.

The antinociceptive effects 134 extracts prepared from 45 species of mushrooms were examined by the acetic acid-induced writhing method. From the CH2Cl2 extract of Ganoderma lucidum among the active extracts, ganoderic acids A, B, G and H and compound C6 were isolated as the antinociceptive components.

Role of nitric oxide towards vasodilator effects of substance P and ATP in human forearm vessels.

1. It has been shown in animals that substance P as well as acetylcholine releases endothelium-derived nitric oxide and evokes vasodilatation and that ATP-induced vasodilatation is partially mediated by nitric oxide. The aim of this study was to examine whether vasodilator effects of substance P and ATP are mediated by nitric oxide in humans. 2. In healthy volunteers (n = 35), we measured forearm blood flow by a strain-gauge plethysmograph while infusing graded doses of acetylcholine, substance P, ATP or sodium nitroprusside into the brachial artery before and after infusion of NG-monomethyl-L-arginine (4 or 8 mumol/min for 5 min). In addition, we measured forearm blood flow while infusing substance P before and during infusion of L-arginine (10 mg/min, simultaneously), or before and 1 h after oral administration of indomethacin (75 mg). 3. Acetylcholine, substance P, ATP or sodium nitroprusside increased forearm blood flow in a dose-dependent manner. NG-Monomethyl-L-arginine decreased basal forearm blood flow and inhibited acetylcholine-induced vasodilatation but did not affect substance P-, ATP-, or sodium nitroprusside-induced vasodilatation. Neither supplementation of L-arginine nor pretreatment with indomethacin affected substance P-induced vasodilatation. 4. Our results suggest that, in the human forearm vessels, substance P-induced vasodilatation may not be mediated by either nitric oxide or prostaglandins and that ATP-induced vasodilatation may also not be mediated by nitric oxide.

Anti-emetic principles of Inula linariaefolia flowers and Forsythia suspensa fruits.

The anti-emetic effects of 40 extracts made from 12 traditional Chinese herbal drugs were examined. Ten extracts inhibited emesis induced by copper sulfate pentahydrate; all were administered orally, and one extract inhibited emesis induced by apomorphine hydrochloride given to leopard and ranid frogs. Taraxasteryl palmitate and acetate, bigelovin and dihydrobigelovin were isolated from the CHCl(3) extract of Inula linariaefolia flowers, and identified as the active antiemetic agents when emesis was induced by copper sulfate. In addition, chlorogenic acid was isolated from the MeOH extract as an anti-emetic principle for the emesis induced by apomorphine hydrochloride. Rengyol, phillyrin and rutin were isolated from the MeOH extract of Forsythia suspensa fruits and identified as the inhibitors of emesis induced by copper sulfate pentahydrate.

Effects on health of dietary supplementation with 100 mg d-alpha-tocopheryl acetate, daily for 6 years.

To evaluate the clinical antioxidant effects of vitamin E, 161 healthy volunteers aged 39 to 56 years, were given 100 or 3 mg of d-alpha-tocopheryl acetate orally daily for 6 years using a randomized, double-blind design. Among the 147 volunteers who qualified for the analysis, seven of the 73 volunteers receiving 3 mg d-alpha-tocopheryl acetate daily and none of the 74 volunteers receiving 100 mg had coronary disorders including myocardial damage (P < 0.02). ST or T wave abnormalities on electrocardiograms were considered to indicate coronary disorders (four volunteers). The mean serum total tocopherol (TOC) concentration in the 100-mg group was significantly higher than that in the 3-mg group 6 months after the start of the study, and this raised value was maintained throughout the study; the level in the 3-mg group did not change significantly from the baseline value. The low-density lipoprotein cholesterol/total TOC ratio, a parameter of the inhibition of peroxidation of low-density lipoprotein cholesterol, was the only serum lipid parameter that was significantly different, at baseline, in the volunteers with coronary disorders compared with the others. These findings indicate that long-term supplementation with 100 mg tocopheryl acetate daily may prevent the early stages of coronary atherosclerosis by decreasing peroxidation of low-density lipoprotein cholesterol.

Relationship of fat deposition and intestinal microflora in VMH rats.

The purpose of this study was to investigate the pathophysiology of the intestinal microflora in the mechanism of fat deposition in hypothalamic obese (VMH) rats. Enterococci of VMH rats decreased 2 weeks after bilateral lesions in the ventromedial hypothalamic nuclei, and significantly decreased at 12 weeks after the lesions were made (P less than 0.01). Lactobacilli increased after 1 week after the lesions. Numbers of enterococci were negatively correlated with fat deposition in the parametrium (P less than 0.01), retroperitoneum (P less than 0.01), and liver (P less than 0.05), and also with Lee's index (P less than 0.02). Lactobacilli were positively correlated with serum glucose (P less than 0.01), fat deposition in the parametrium (P less than 0.02), and Lee's index (P less than 0.05). Enterococci probably decrease the intestinal absorption of cholesterol, and lactobacilli facilitate the absorption of hexose in the intestine, so a decrease in enterococci and increase in lactobacilli caused by hypothalamic lesions may accelerate the pathogenesis of obesity in VMH rats.