Miniaturized glucose biosensor modified with a nitric oxide-releasing xerogel microarray

An enzyme-based glucose biosensor modified to release nitric oxide (NO) via a xerogel microarray is reported. The biosensor design is as follows: (1) glucose oxidase (GOx) is immobilized in a methyltrimethoxysilane (MTMOS) xerogel layer; (2) a blended polyurethane/hydrophilic polyurethane coating prevents enzyme leaching and imparts selectivity for glucose; and (3) micropatterned xerogel lines (5 microm wide) separated by distances of 5 or 20 microm provide NO-release capability. This configuration allows for increased glucose sensitivity relative to sensors modified with NO-releasing xerogel films since significant portions of the sensor surface remain unmodified. Glucose diffusion to the GOx layer is thus less inhibited. The micropatterned NO-releasing biosensors generate sufficient NO levels to reduce both Pseudomonas aeruginosa and platelet adhesion without significantly compromising the enzymatic activity of GOx. The glucose response, linearity and stability of the NO-releasing micropatterned sensors are reported.     

Nitric oxide-releasing hydrophobic polymers: preparation, characterization, and potential biomedical applications

The synthetic methods used recently in this laboratory to prepare a variety of novel nitric oxide (NO)-releasing hydrophobic polymers are reviewed. Nitric oxide is a well known inhibitor of platelet adhesion and activation. Thus, such NO release polymers have potential applications as thromboresistant coatings for a large number of blood-contacting biomedical devices (e.g., in vivo sensors, arteriovenous grafts, stents, catheters, extracorporeal circuits). The approaches taken to prepare NO releasing poly(vinyl chloride) (PVC), silicone rubber (SR), polymethacrylate (PM), and polyurethane (PU) materials are grouped into three categories: (1) dispersion/doping of discrete diazeniumdiolated molecules within the polymeric films; (2) chemical derivatization of polymeric filler microparticles (e.g., silicon dioxide, titanium dioxide) to possess NO release chemistry and then their dispersion within the hydrophobic polymers; and (3) covalent attachment of NO release moieties to polymer backbones. Specific chemical examples of each of these approaches are summarized and the advantages and disadvantages of each are discussed. Other related work in the field of NO release polymers is also cited. It is further shown that several of the NO-releasing polymeric materials already prepared exhibit the expected improved thromboresistivity when tested in vivo using appropriate animal models.     

Efficacy of surface-generated nitric oxide against Candida albicans adhesion and biofilm formation

This report details the efficacy of nitric oxide (NO)-releasing xerogel surfaces composed of N-(6-aminohexyl)aminopropyl trimethoxysilane (AHAP3) and isobutyltrimethoxysilane (BTMOS) against Candida albicans adhesion, viability, and biofilm formation. A parallel plate flow cell assay was used to examine the effect of NO on planktonic fungal cells. Nitric oxide fluxes as low as 14 pmol cm^?2 s^?1 were sufficient to reduce fungal adhesion by ～49% over the controls after 90 min. By utilizing a fluorescence live/dead assay and replicate plating, NO flux was determined to reduce fungal viability in a dose-dependent manner. The formation of C. albicans biofilms on NO-releasing xerogel-coated silicon rubber (SiR) coupons was impeded when compared to control (non-NO-releasing) and bare SiR surfaces. The synergistic efficacy of NO and silver sulfadiazine against adhered fungal cells and biofilms is reported with increased killing and biofilm inhibition over NO alone.     

Poly(vinylpyrrolidone)-doped nitric oxide-releasing xerogels as glucose biosensor membranes

Nitric oxide (NO)-releasing xerogel membranes were prepared as coatings for an electrochemical glucose biosensor to allow for enhanced biocompatibility while maintaining adequate response times and sensitivity. Formation of the NO-donor species was found to drastically decrease the permeability of the aminosilane-based xerogels to both hydrogen peroxide and glucose. The addition of poly(vinylpyrrolidone) (PVP) polymer enhanced the membrane permeability even after exposure to high pressures of NO (necessary for NO-donor synthesis). The analytical response and NO release of PVP-doped NO-releasing xerogels as glucose sensor membranes were further investigated and found to be enhanced via polymer doping. Doping of the polymer into the xerogel did not compromise the stability of the xerogel as evaluated by silicon leaching studies. Despite the addition of PVP, the NO-releasing xerogels maintained reduced bacterial adhesion characteristics analogous to previous reports for NO-releasing xerogels.     

The effects of nitric oxide in settlement and adhesion of zoospores of the green alga Ulva

Previous studies have shown that elevated nitric oxide (NO) reduces adhesion in diatom, bacterial and animal cells. This article reports experiments designed to investigate whether elevated NO reduces the adhesion of zoospores of the green alga Ulva, an important fouling species. Surface-normalised values of NO were measured using the fluorescent indicator DAF-FM DA and parallel hydrodynamic measurements of adhesion strength were made. Elevated levels of NO caused by the addition of the exogenous NO donor SNAP reduced spore settlement by 20% and resulted in lower adhesion strength. Addition of the NO scavenger cPTIO abolished the effects of SNAP on adhesion. The strength of attachment and NO production by spores in response to four coatings (Silastic® T2; Intersleek® 700; Intersleek® 900 and polyurethane) shows that reduced adhesion is correlated with an increase in NO production. It is proposed that in spores of Ulva, NO is used as an intracellular signalling molecule to detect how conducive a surface is for settlement and adhesion. The effect of NO on the adhesion of a range of organisms suggests that NO-releasing coatings could have the potential to control fouling.     

Nitric oxide as a regulator of inflammatory processes

Nitric oxide (NO) plays an important role in mediating many aspects of inflammatory responses. NO is an effector molecule of cellular injury, and can act as an anti-oxidant. It can modulate the release of various inflammatory mediators from a wide range of cells participating in inflammatory responses (e.g., leukocytes, macrophages, mast cells, endothelial cells, and platelets). It can modulate blood flow, adhesion of leukocytes to the vascular endothelium and the activity of numerous enzymes, all of which can have an impact on inflammatory responses. In recent years, NO-releasing drugs have been developed, usually as derivatives of other drugs, which exhibit very powerful anti-inflammatory effects.     

一株奇异变形杆菌对铜绿假单胞菌多细胞行为的抑制作用

目的：抗生素耐药性成为了全球性的健康问题。研究发现病原菌的多细胞行为在抗生素的耐药性中起着至关重要的作用（尤其是生物膜）,因而通过抑制多细胞行为而控制耐药性成为当务之急。本文以奇异变形杆菌（Proteus mirabilis）为研究对象,考察它的发酵滤液对一种机会致病菌——铜绿假单胞菌（Pseudomonas aeruginosa）多细胞行为的作用,以期得到一株多细胞行为抑制菌：在不影响Paerugiliosa生长的前提下,抑制生物膜形成、EPS产生以及定向丛集运动,解除保护,减缓扩散,为降低Paemgi—nosa耐药性,增强抗生素作用效果提供可能。方法：采用结晶紫生物膜测定法、蒽酮一硫酸法、平板检测法,探究Pmirabilis发酵滤液对Paemginosa生物膜、胞外多聚物、定向丛集运动和生长的影响。结果：Pmirabilis发酵滤液能显著抑制Paeruginosa生物膜量,在体积百分比浓度为1％时,抑制率可达60．9％。该菌的发酵滤液还能阻碍Paeruginosa的定向丛集运动,减弱它的吸附和扩散运动;同时,也减少了Pacrugillosa胞外多聚物的产量,在滤液体积百分比浓度为1％时,抑制率达到45．9％。更重要的是,固体平板实验证明该发酵滤液对P．aemginosa的生长没有影响。结论：Pmirabilis在不影响病原菌生长的前提下,对病原菌的多细胞行为有一定的控制作用。其发酵滤液中存在着抑制微生物膜、定向丛集运动等的成分,在治疗细菌感染性疾病和降低抗生素耐药性方面有潜在应用价值。     

N-hydroxyl derivatives of guanidine based drugs as enzymatic NO donors

Recent research suggests that NO may play a role in the physiological effects of some guanidine-containing drugs. In this report, three guanidine-containing drugs (guanadrel, guanoxan, and guanethidine) together with their N-hydroxyl derivatives were synthesized and their NO-releasing abilities catalyzed by nitric oxide synthases (NOSs) and horseradish peroxidase were evaluated. The guanidine containing compounds could not release NO in the presence of NOS or peroxidase. The corresponding N-hydroxyl compounds exhibited weak NO-releasing ability under the catalyzed of NOS and good NO-releasing ability under the oxidation by horseradish peroxidase in the presence of H(2)O(2). These compounds also displayed vasodilatory activity.     

Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay

Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70?% and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2)?cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100?% of E. coli, P. aeruginosa, P. mirabilis and 95?% of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3?days for detection.     

Activity of nitric oxide is dependent on, but is partially required for function of, salicylic acid in the signaling pathway in tobacco systemic acquired resistance.

When tobacco plants were treated by injection with nitric oxide (NO)-releasing compounds, the sizes of lesions caused by Tobacco mosaic virus (TMV) on the treated leaves and on upper nontreated leaves were significantly reduced. The reduction in TMV lesion size was caused by NO released from the NO-releasing compounds; the byproduct formed after release of NO from the NO-releasing compound NOC-18, diethylenetriamine, did not itself alter lesion size. Treatment of tobacco plants with inhibitors of nitric oxide synthase or an NO scavenger attenuated but did not abolish the systemic acquired resistance (SAR) induced by salicylic acid (SA). In NahG transgenic tobacco plants, NO had no effect on lesion size following TMV infection. These results are consistent with the hypothesis that NO plays an important role in SAR induction in tobacco and that NO is required for the full function of SA as an SAR inducer. The activity of NO is fully dependent on the function of SA in the SAR signaling pathway in tobacco.     

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats

Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5'-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation. Treatment of porcine PA endothelial cells (PAEC) with P1 caused time-dependent increase in intracellular NO release and inhibition of the catalytic activity of cGMP-specific PDE-5 but not PDE-5 protein expression leading to increased levels of cGMP. Acute hypoxia-induced PA vasoconstriction ex vivo and continuous telemetry monitoring of hypoxia (10% oxygen)-induced elevated PA pressure in freely moving rats were significantly restored by administration of P1. Chronic hypoxia (10% oxygen for 4 weeks)-induced alterations in PA perfusion pressure, right ventricular hypertrophy, and vascular remodeling were attenuated by P1 treatment. These results demonstrate the potential therapeutic effects of P1 to prevent and/or arrest the progression of hypoxia-induced PAH via NO/cGMP-dependent modulation of hemodynamic and vascular remodeling in the pulmonary circulation.     

Kinase suppressor of Ras-1 protects against pulmonary Pseudomonas aeruginosa infections

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe infections in immunocompromised individuals and individuals with cystic fibrosis or chronic obstructive pulmonary disease (COPD). Here we show that kinase suppressor of Ras-1 (Ksr1)-deficient mice are highly susceptible to pulmonary P. aeruginosa infection accompanied by uncontrolled pulmonary cytokine release, sepsis and death, whereas wild-type mice clear the infection. Ksr1 recruits and assembles inducible nitric oxide (NO) synthase (iNOS) and heat shock protein-90 (Hsp90) to enhance iNOS activity and to release NO upon infection. Ksr1 deficiency prevents lung alveolar macrophages and neutrophils from activating iNOS, producing NO and killing bacteria. Restoring NO production restores the bactericidal capability of Ksr1-deficient lung alveolar macrophages and neutrophils and rescues Ksr1-deficient mice from P. aeruginosa infection. Our findings suggest that Ksr1 functions as a previously unknown scaffold that enhances iNOS activity and is therefore crucial for the pulmonary response to P. aeruginosa infections.     

Fabrication of nitric oxide-releasing polyurethane glucose sensor membranes

Despite clear evidence that polymeric nitric oxide (NO) release coatings reduce the foreign body response (FBR) and may thus improve the analytical performance of in vivo continuous glucose monitoring devices when used as sensor membranes, the compatibility of the NO release chemistry with that required for enzymatic glucose sensing remains unclear. Herein, we describe the fabrication and characterization of NO-releasing polyurethane sensor membranes using NO donor-modified silica vehicles embedded within the polymer. In addition to demonstrating tunable NO release as a function of the NO donor silica scaffold and polymer compositions and concentrations, we describe the impact of the NO release vehicle and its release kinetics on glucose sensor performance.     

Nitric oxide-producing polyurethanes

Thrombus formation and eventual intimal hyperplasia are the leading causes of small-diameter synthetic vascular graft failure. To combat these issues, we have incorporated a diazeniumdiolate-modified nitric oxide (NO)-producing peptide into a polyurethane to improve the thromboresistance of this biocompatible polymer. NO production by polyurethane films occurred for approximately 2 months under physiological conditions, and mechanical properties of the material were suitable for vascular graft applications. Platelet adhesion to NO-releasing polyurethane was dramatically decreased compared to control polyurethane. Furthermore, endothelial cell growth was stimulated in the presence of the NO-releasing polyurethane, while smooth muscle cell growth was greatly inhibited. The ability of this bioactive material to inhibit platelet adhesion and smooth muscle cell proliferation while encouraging endothelialization suggests that this NO-generating polyurethane may be suitable as a candidate material for small-diameter vascular grafts.     

The aqueous garlic, onion and leek extracts release nitric oxide from S-nitrosoglutathione and prolong relaxation of aortic rings

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys > GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 μmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.     

The binding of Proteus mirabilis nonagglutinating fimbriae to ganglio-series asialoglycolipids and lactosyl ceramide

Proteus mirabilis is a common opportunistic Gram-negative uropathogen that infects the upper urinary tract. We have examined the role of the nonagglutinating fimbriae (NAF) of P. mirabilis in mediating bacterial adhesion to cell surface receptors. Purified NAF of P. mirabilis were demonstrated to bind to a number of glycolipids, including asialo-GM1, asialo-GM2, and lactosyl ceramide (LacCer) in solid-phase binding assays and in thin layer chromatography (TLC) overlay assays. Furthermore, preincubation of the biotinylated NAF (Bt-NAF) with anti-NAF monoclonal antibodies resulted in inhibition of NAF binding to immobilized asialo-GM1, asialo-GM2, and LacCer. In adherence assays, P. mirabilis binding to Madin-Darby canine kidney (MDCK) cells was inhibited by murine anti-asialo-GM1 monoclonal antibodies H2G10 to about 50% of the binding level in the absence of the antibody, specific for the terminal beta-galactopyranosyl residue of asialo-GM1 (Harrison et al. 1998). The results of this study suggest that NAF of P. mirabilis recognize a GalNAc beta 1-4Gal moiety present in the ganglio-series of asialoglycolipids, and that the terminal beta-galactopyranosyl-containing glycoconjugates play a role in NAF-mediated adherence of P. mirabilis to MDCK cells. Similarly to other bacteria, P. mirabilis NAF was also shown to express the LacCer specificity.     

一株奇异变形杆菌对铜绿假单胞菌多细胞行为的抑制作用*

摘要目的：抗生素耐药性成为了全球性的健康问题。研究发现病原菌的多细胞行为在抗生素的耐药性中起着至关重要的作用 （尤其是生物膜）,因而通过抑制多细胞行为而控制耐药性成为当务之急。本文以奇异变形杆菌（Proteus Mirabilis ）为研究对象,考 察它的发酵滤液对一种机会致病菌———铜绿假单胞菌（ Pseudomonas aeruginose）多细胞行为的作用,以期得到一株多细胞行为抑 制菌：在不影响 P.aeruginosa 生长的前提下,抑制生物膜形成、EPS 产生以及定向丛集运动,解除保护,减缓扩散,为降低P.aeruginosa 耐药性,增强抗生素作用效果提供可能。方法：采用结晶紫生物膜测定法、蒽酮-硫酸法、平板检测法,探究P.aeruginosa 发酵滤 液对P.aeruginosa 生物膜、胞外多聚物、定向丛集运动和生长的影响。结果： P.aeruginosa 发酵滤液能显著抑制生物膜 量,在体积百分比浓度为1 %时,抑制率可达60.9 %。该菌的发酵滤液还能阻碍的定向丛集运动,减弱它的吸附和扩 散运动;同时,也减少了P.aeruginosa 胞外多聚物的产量,在滤液体积百分比浓度为1 %时,抑制率达到45.9%。更重要的是,固体 平板实验证明该发酵滤液对P.aeruginosa 的生长没有影响。结论： 在不影响病原菌生长的前提下,对病原菌的多细胞 行为有一定的控制作用。其发酵滤液中存在着抑制微生物膜、定向丛集运动等的成分,在治疗细菌感染性疾病和降低抗生素耐药 性方面有潜在应用价值。     

The effect of aspirin and two nitric oxide donors on the infarcted heart in situ

Nitric oxide (NO) donors are heterogeneous substances which release NO, a biologically active compound. NO released by nitric oxide donors has important effects on the circulation by causing vasodilation, diminishing myocardial contractile force, inhibiting platelet aggregation, and counteracting the effects of thromboxane A2. In the infarcted heart, activation of the inducible form of nitric oxide synthase (iNOS) and the formation of prostacyclin and thromboxane A2 by cyclooxygenase (COX) were increased. Myocardial infarction also resulted in increased myocardial NO production. Aspirin (acetylsalicylic acid. ASA) at low concentration (35 mg/kg/day) fails to change iNOS production, in contrast to higher dose (150 mg/kg/day) which, as previously shown, inhibits iNOS activity. ASA at all doses also suppresses myocardial prostanoid formation because of inhibition of COX. Recently, two NO donors have been synthesized: NCX 4016 and Diethylenetriamine/NO (DETA/NO). NCX 4016 combines an NO-releasing moiety with a carboxylic residue via an esteric bond. We describe here that NCX 4016 (65 mg/kg/day) increased prostacyclin and thromboxane A2 production in the infarcted heart muscle, overcoming the inhibitory effects of ASA. As a result of nitric oxide release, oxidation products of NO (NO2- and NO3-; NOx) in arterial blood rose following administration of NCX 4016. On oral administration, NCX 4016 did not change systemic arterial pressure. The effects of a single NO donor, DETA/NO (1.0 mg/kg/day) on the infarcted heart were also investigated On intravenous administration, the compound increased NO concentration in arterial blood slightly but to a lesser degree than NCX 4016. Like NCX 4016, it raised myocardial production of prostacyclin and thromboxane A2 in the infarcted heart. However, it caused a severe fall in blood pressure. These findings demonstrate that newly-synthesized NO donors release nitric oxide in situ and increase myocardial production of prostanoids. NCX 4016 has therapeutic potential because it can be orally administered, lacks hypotensive effects, increases blood levels of nitric oxide and myocardial prostacyclin production.     

Modulation of neuronal nitric oxide release by soluble guanylyl cyclase in guinea pig colon

Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexus-containing smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO(-)(2). In the presence of 8-Br-cGMP, YC-1 and zaprinast (all at 10(-4) M) the NO/NO(-)(2)-release increased to 152 +/- 16% (P < 0.05), 164 +/- 37% (P < 0.05) and 290 +/- 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10(-5) M) decreased the evoked release of NO/NO(-)(2) to 49 +/- 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones.