Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a deltamexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a deltamexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including deltamexB and deltamexXY mutants, were intermediate between WT and the deltamexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the deltamexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.     

CGRP and PACAP-38 play an important role in diagnosing pediatric migraine

BackgroundAn increasing number of studies have suggested that the important role of vasoactive peptides, such as pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and calcitonin gene-related peptide (CGRP), in the pathophysiology of migraine seems undeniable in adults, but studies in pediatric migraine patients remain scarce. We prospectively investigated CGRP and PACAP-38 plasma levels in children with migraine during ictal and interictal periods and compared the results between migraine patients with aura and without aura. We were the first to explore the diagnostic value of a combination of CGRP and PACAP-38.MethodsSeventy-six migraine patients aged 4–18 years and seventy-seven age-matched healthy children were included in the study. Plasma vasoactive peptides were measured using the enzyme-linked immunosorbent assay (ELISA). Differences and correlations of groups were analyzed using the independent samples t-test, analysis of variance (ANOVA), Mann-Whitney U test, and multiple linear regression. We also performed logistic regression and receiver operating characteristic curve (ROC) analyses to evaluate the diagnostic value of CGRP and PACAP-38 in pediatric migraine.ResultsPACAP-38 and CGRP levels in migraine patients during the ictal and interictal periods were higher than those in controls (p < 0.001). PACAP-38 and CGRP levels in migraine patients with aura and without aura were higher than those in controls (p < 0.001). PACAP-38 and CGRP were independent risk factors in diagnosing pediatric migraine (adjusted OR (PACAP-38) =1.331, 95% CI: 1.177–1.506, p < 0.001; adjusted OR (CGRP) = 1.113, 95% CI: 1.064–1.165, p < 0.001). Area Under Curve (AUC) comparison: Combination (0.926) > CGRP (0.869) > PACAP-38 (0.867).ConclusionsOur study found almost the same changes in CGRP and PACAP levels in pediatric migraine, suggesting that CGRP and PACAP-38 may work together to play an integral role in pediatric migraine. Higher CGRP levels were found in the ictal phase than in the interictal phase and with aura group than without aura group, indicating that CGRP may take part in the formation of pain and aura. Moreover, ROC and logistic regression analyses suggested that CGRP and PACAP-38 are good indicators to diagnose pediatric migraine, and the combination of CGRP and PACAP-38 was valuable in diagnosing pediatric migraine and differentiating pediatric migraine from non-migraine headaches.Trial registrationThe study has been registered at the Chinese Clinical Trial Registry (ChiCTR2100043157).     

构建内皮型一氧化氮合酶真核表达载体及在内皮细胞的表达

背景:内皮型一氧化氮合酶表达产物对内皮细胞的影响是血管外科基础研究中的重要课题,对于生物人工血管的研制有重要意义。目的:构建内皮型一氧化氮合酶基因真核表达载体pcDNA3.1/eNOS,以脂质体为介导观察其在血管内皮细胞中的转染效率,评价表达产物内皮型一氧化氮合酶的酶学活性及其对内皮细胞生长的影响。设计、时间及地点:单一样本观察,实验于2005-07/2007-05在南京大学医学院附属鼓楼医院生物化学实验室完成。材料:质粒PCMV-eNOS由勋阳医学院张群林教授惠赠。pcDNA3.1真核表达载体由南京大学博士后贾立军馈赠,pcDNA3.0/EGFP质粒由戚金亮博士提供。大肠杆菌DH5a和人脐静脉内皮细胞株(ECV304)为南京大学生物系保存。方法:采用分子生物学技术,先用限制性核酸内切酶EcorⅠ酶切质粒PCMV/eNOS,电泳回收3.6kb编码人内皮型一氧化氮合酶cDNA序列,克隆入质粒pcDNA3.1的EcorⅠ酶切位点,通过内皮型一氧化氮合酶cDNA序列中的XhoⅠ酶切位点筛选其插入载体的方向,构建pcDNA3.1/eNOS重组质粒。以Lipofec-tamine为介导将其与pcDNA3.0/EGFP共转染分离培养的人脐静脉内皮细胞(ECV304)。主要观察指标:①观察转染后对ECV304生长的影响,在流式细胞仪和荧光显微镜下计算转染效率。②以反转录-聚合酶链反应和免疫组织化学检测内皮型一氧化氮合酶基因的表达,测定内皮型一氧化氮合酶活性以及一氧化氮产量。③观察施加不同因素(L-精氨酸、氯化钙、乙二胺四乙酸、L-NAME)对内皮型一氧化氮合酶活性及一氧化氮合成的影响;同时检测此时ECV304生长所受影响。结果:以脂质体为介导转染人脐静脉ECV304后,其转染效率为(32.6±2.6)%,反转录-聚合酶链反应和免疫组织化学检测到相应的内皮型一氧化氮合酶mRNA和蛋白表达,与对照组有明显差别;同时在上述不同因素影响下内皮型一氧化氮合酶活性出现明显差别;随着培养液一氧化氮浓度升高,ECV304生长曲线下降。结论:成功构建pcDNA3.1/eNOS,在脂质体介导下能高效转染ECV304并良好表达,Ca2 是内皮型一氧化氮合酶的必要激活条件,ECV304的增生受到高浓度一氧化氮抑制。     

The role of nitric oxide in nociception

Pharmacologic, electrophysiologic, and immunohistochemical studies have suggested a role of nitric oxide (NO) in nociception processing. Recent studies have indicated that NO may modulate spinal and sensory neuron excitability through multiple mechanisms that may underlie its distinctive roles in different pain states. Differential regulation of a family of NO-producing enzymes, NO synthases, contributes mainly to the complexity underlying the role of NO in nociception. This review summarizes the latest advances in our understanding of the contribution of NO to pain transduction. Possible cellular mechanisms regarding the connection between NO production and the abnormal sensation derived from different stimuli and pathologic conditions are discussed.     

The role of nitric oxide synthase/nitric oxide in vascular smooth muscle control

Vascular compliance is dependent on endogenous and exogenous sources of nitric oxide (NO). In a discussion of therapeutics and NO derived via nitric oxide synthase (NOS) enzymes, it is necessary to examine the pathways of each drug to provide the clinical perfusionist with a greater understanding of the role of NOS/NO in vascular function. Endothelial-derived NO is a contributor in the vasoregulation of vascular smooth muscle. Therapeutics seek to mimic the vasodilatory effects of the endogenous NO. The therapeutics included in this review are nitroglycerin, nitroprusside, amyl nitrite, and inhalation of NO. L-Arginine supplementation provides additional substrate for the endogenous pathway that can augment NO production. NO is a small bioactive molecule involved in various biochemical pathways. Dysregulation of NO production can impair normal physiologic control of vascular compliance. Therefore, the purpose of this review is to provide the perfusionist with an understanding of the biochemical and pharmacological aspects of NOS/NO associated with vascular function.     

Immune dysfunction in endotoxicosis: role of nitric oxide produced by inducible nitric oxide synthase

OBJECTIVE: To investigate whether stimulation with lipopolysaccharide (LPS) alters cytokine production by splenocytes in mice and whether it changes the T-helper 1 (Th1)/Th2 balance. The role of nitric oxide in such immunologic changes was also explored using mice with genetic lack of inducible nitric oxide synthase (iNOS). DESIGN:: Prospective animal study with concurrent controls. SETTING: University research laboratory. SUBJECTS: iNOS knockout mice and wild-type littermates. INTERVENTIONS: iNOS knockout mice or wild-type mice were injected with LPS or saline with or without anti-interleukin (IL)-6 antibody, and survival was monitored for 7 days. MEASUREMENTS AND MAIN RESULTS: At 24 and 48 hrs after administration, blood samples and splenocytes were obtained to examine immunologic variables. Cell surface markers and cytokine expression of splenocytes were used to characterize the Th1/Th2 balance and were measured by flow cytometry. At 48 hrs after LPS administration, the Th1/Th2 balance shifted toward Th2 predominance in wild-type mice, irrespective of the IL-6 level, whereas it showed Th1 predominance in iNOS knockout mice, and the increase of IL-6 and IL-10 in response to LPS persisted in these animals. After LPS administration, the mortality rate was significantly higher in iNOS knockout mice than in wild-type mice, irrespective of the IL-6 level. CONCLUSIONS: These findings suggest that nitric oxide produced by iNOS during endotoxemia may be involved in down-regulation of Th1 cytokines and up-regulation of Th2 cytokines, whereas IL-6 has no such role. The increased lethality of LPS in iNOS knockout mice suggests that nitric oxide may be protective against proinflammatory cytokine-induced damage. Nitric oxide excess may increase susceptibility to nosocomial infections, so-called immunoparalysis.     

Endothelial nitric oxide contributes to the renal protective effects of ischemic preconditioning

We determined whether endothelial nitric oxide synthase (eNOS) plays an important role in the renal protective effect of ischemic preconditioning (IP) against the ischemia/reperfusion-induced acute renal failure (ARF) by using eNOS-deficient (eNOS(-/-)) and wild-type (eNOS(+/+)) mice. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. IP, which consists of three cycles of 2-min ischemia followed by 5-min reperfusion, was performed prior to 45-min ischemia. In eNOS(+/+) mice, IP treatment markedly attenuated the ischemia/reperfusion-induced renal dysfunction and significantly improved histological renal damage such as tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. Constitutive nitric oxide synthase activity in the kidney without IP was markedly decreased 6 h after reperfusion, but this decreased response was not observed in eNOS(+/+) mice with IP treatment. The improvement of renal dysfunction in eNOS(+/+) mice with IP treatment was abolished by pretreatment with N(G)-nitro-l-arginine, a nonselective NOS inhibitor, whereas aminoguanidine, an inducible NOS inhibitor, had no effect. Finally, no protective effects of IP on ischemia/reperfusion-induced renal dysfunction and histological damage were observed in eNOS(-/-) mice. These findings strongly support the view that eNOS-mediated NO production plays a pivotal role in the protective effect of IP on ischemia/reperfusion-induced ARF.     

The role of nitric oxide in the post-ischemic revascularization process

Following arterial occlusion, blood vessels respond by sprouting new capillaries (i.e. angiogenesis) and by growing and remodelling pre-existing arterioles into physiologically relevant arteries (i.e. arteriogenesis). The importance of nitric oxide (NO) in ischemia-induced angiogenesis is supported by 4 main findings: (i) the ischemic limb shows an increase in endothelial nitric oxide synthase (eNOS) mRNA, protein expression and NO synthesis; (ii) the absence of the NO pathway (by either pharmacological inhibition or gene disruption of eNOS) abolishes ischemia-induced angiogenesis; (iii) supplementation of NO by the use of exogenous sources restores ischemia-induced angiogenesis; and (iv) cardiovascular diseases associated with decreased NO synthesis show impaired ischemia-induced angiogenesis. Thus, impairment of the NO metabolic pathway could be one of the main contributing factors for the development of peripheral arterial occlusive disease. The restoration of normal NO levels in diseased arteries is therefore a major therapeutic goal; this could be achieved by supplementation with exogenous NO or by strategies designed to increase the concentration of endogenous NO.     

Cocaine-mediated apoptosis in bovine coronary artery endothelial cells: role of nitric oxide.

The present study examined the role of nitric oxide in cocaine-induced apoptosis in bovine coronary artery endothelial cells (BCAECs). Cocaine produced a time-dependent decrease in cell viability and an increase in apoptosis in BCAECs, which were blocked by the nitric oxide donors DETA-NONOate (DETA-NO) and S-nitroso-N-acetyl-penicillamine. In accordance, cocaine decreased nitric oxide production in BCAECs at each time point of the study. Cocaine significantly increased caspase-3 activity that was blocked by the inhibitors of cytochrome c release (cyclosporin A), caspase-3 (Ac-DEVD-CHO), and caspase-9 (Z-LEHD-FMK), respectively. In addition, cocaine activated caspase-9, which was blocked by cyclosporin A and Z-LEHD-FMK. Ac-DEVD-CHO only partially blocked cocaine-induced caspase-9 activity. DETA-NO (20 microM) blocked cocaine-mediated activation of both caspase-9 and caspase-3. Cocaine decreased Bcl-2 protein levels, which was partially blocked by Ac-DEVD-CHO and Z-LEHD-FMK, but not by DETA-NO. Furthermore, cocaine induced a translocation of Bax from the cytosol to the mitochondria in BCAECs, and increased Bax levels in mitochondria by 2.2-fold. In accordance, cytosolic Bax levels decreased about 42%. Neither Ac-DEVD-CHO nor DETA-NO affected cocaine-induced translocation of Bax. We conclude that cocaine-induced Bcl-2 protein down-regulation and Bax translocation to the mitochondria are upstream signals of caspase-9 activation that precedes caspase-3. Cocaine-induced attenuation of nitric oxide plays a key role in the activation of the caspase cascade in BCAECs.     

Modulation of pain in osteoarthritis: the role of nitric oxide

BACKGROUND: Patients with osteoarthritis (OA) may experience severe pain, progressive loss of movement function, and disability. Many pain-relieving medications are not effective, and are not able to improve the existing pathology. OBJECTIVES: This review summarizes (1) the pathology, mechanisms of pain production, and conservative management of OA with respect to pain; and (2) explains the role of nitric oxide (NO) in pain reduction and production, especially as related to OA. DISCUSSION: NO is produced in biologic cells by a family of enzymes referred to as the nitric oxide synthases (NOSs). The beneficial or harmful effects of different isoforms, constitutive NOS (cNOS) and inducible NOS (iNOS), respectively, suggest dual effects of NO in biologic structures. The harmful effects of NO are most often reported in the literature. We suggest that (1) NO via the beneficial cNOS pathway is decreased in joint structures exposed to chronic load-induced stresses and biochemical change-induced stresses, (2) monochromatic infrared light energy at an 890 nm wavelength, applied at the skin surface, is absorbed into blood vessels and stimulates production of NO in joints by the beneficial cNOS pathway, (3) NO from the cNOS pathway may help decrease the detrimental effects of NO induced by iNOS and produced in OA pathology, and (4) NO-based intervention may produce substantial pain relief without undesirable side effects by increasing circulation, decreasing nerve irritation, and decreasing inflammation in joints. KEY MESSAGES: (1) The roles of NO in nociception are dual and complex. (2) NO via cNOS, produced transiently in small amounts, can bring dramatic relief to people with painful OA.     

Anticoagulative effect of nitric oxide inhalation in ARDS

Some studies have suggested that nitric oxide (NO) may cause platelet dysfunction. We present an ARDS patient who need this treatment, with a transient alteration of platelet function and a significant prolongation of bleeding time. Received: 2 July 1997 Accepted: 24 April 1998     

Inducible nitric oxide synthase in rat hepatic lipocytes and the effect of nitric oxide on lipocyte contractility.

In liver injury, perisinusoidal cells known as lipocytes (Ito cells) undergo "activation," acquiring smooth muscle-like features and a contractile phenotype. To assess whether contraction of these cells is regulated by nitric oxide (NO), we examined the production of NO by lipocytes and the effect of NO on lipocyte contractility. Cultured lipocytes were exposed to cytokines and/or LPS. Single agents had little or no effect on the level of inducible NO synthase (iNOS) mRNA. However, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), or LPS in combination with interferon-gamma (IFN-gamma) stimulated iNOS mRNA, which was present within 4 h after exposure. iNOS mRNA levels were paralleled by changes in nitrite (a metabolic product of NO). Intraperitoneal administration of IFN-gamma, TNF-alpha, and LPS led to rapid induction of iNOS mRNA in lipocytes, confirming in vivo the culture findings. Ligation of the common hepatic bile duct, which induces periportal-based liver injury, stimulated iNOS mRNA in lipocytes. Transforming growth factor-beta 1 decreased IFN-gamma/TNF-alpha--stimulated iNOS mRNA and nitrite. Finally, the effect of NO on lipocyte contractility was examined. In cells incubated with IFN-gamma and TNF-alpha, the contractile response to either serum or endothelin-1 was blocked. Contraction was restored entirely by an inhibitor of NO synthase, NG-monomethylarginine. Furthermore, 8-bromoguanosine 3':5'-cyclic monophosphate and sodium nitroprusside inhibited lipocyte contractility, consistent with the effect of NO induced by cytokines. We conclude that NO is a potent modulator of lipocyte contractility and may regulate this function by autocrine (or intracrine) mechanisms. Moreover, NO may play an important role in liver injury, countering the effect of contractile agonists on lipocytes.