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Controlling platelet activation and clotting initiated by cardiovascular interventions remains a major challenge in clinical practice. In this work, the anti-thrombotic properties of a polysaccharide-based nitric oxide (NO)-releasing dextran derivative are presented. Total platelet adhesion, platelet morphology and whole blood clotting kinetics were used as indicators to evaluate the anti-clotting properties of this material. With a total NO delivery of 0.203±0.003 μmol, the NO-releasing dextran derivative (Dex-SNO) mixed with blood plasma demonstrated a significantly lower amount of platelet adhesion and activation onto a surface and reduced whole blood clotting kinetics. Nearly 75% reduction in platelet adhesion and a significant retention of platelet morphology were observed with blood plasma treated with Dex-SNO, suggesting this to be a potential anti-platelet therapeutic agent for preventing thrombosis that does not have an adverse effect on circulating platelets.

Coding a wide range of light intensities in natural scenes poses a challenge for the retina: adaptation to bright light should not compromise sensitivity to dim light. Here we report a novel form of activity-dependent synaptic plasticity, specifically, a "weighted potentiation" that selectively increases output of Mb-type bipolar cells in the goldfish retina in response to weak inputs but leaves the input-output ratio for strong stimuli unaffected. In retinal slice preparation, strong depolarization of bipolar terminals significantly lowered the threshold for calcium spike initiation, which originated from a shift in activation of voltage-gated calcium currents (ICa) to more negative potentials. The process depended upon glutamate-evoked retrograde nitric oxide (NO) signaling as it was eliminated by pretreatment with an NO synthase blocker, TRIM. The NO-dependent ICa modulation was cGMP independent but could be blocked by N-ethylmaleimide (NEM), indicating that NO acted via an S-nitrosylation mechanism. Importantly, the NO action resulted in a weighted potentiation of Mb output in response to small (≤-30 mV) depolarizations. Coincidentally, light flashes with intensity ≥ 2.4 × 10(8) photons/cm(2)/s lowered the latency of scotopic (≤ 2.4 × 10(8) photons/cm(2)/s) light-evoked calcium spikes in Mb axon terminals in an NEM-sensitive manner, but light responses above cone threshold (≥ 3.5 × 10(9) photons/cm(2)/s) were unaltered. Under bright scotopic/mesopic conditions, this novel form of Mb output potentiation selectively amplifies dim retinal inputs at Mb → ganglion cell synapses. We propose that this process might counteract decreases in retinal sensitivity during light adaptation by preventing the loss of visual information carried by dim scotopic signals.

One mechanism of the failure of blood-contacting devices is clotting. Nitric oxide (NO) releasing materials are seen as a viable solution to the mediation of surface clotting by preventing platelet activation; however, NO's involvement in preventing clot formation extends beyond controlling platelet function. In this study, we evaluate NO's effect on factor XII (fibrinogen) adsorption and activation, which causes the initiation of the intrinsic arm of the coagulation cascade. This is done by utilizing a model plasticized poly(vinyl) chloride (PVC), N-diazeniumdiolate system and looking at the adsorption of fibrinogen, an important clotting protein, to these surfaces. The materials have been prepared in such a way to eliminate changes in surface properties between the control (plasticized PVC) and composite (NO-releasing) materials. This allows us to isolate NO release and determine the effect on the adsorption of fibrinogen, to the material surface. Surprisingly, it was found that an NO releasing material with a surface flux of 17.4 ± 0.5 × 10(-10) mol NO cm(-2) min(-1) showed a significant increase in the amount of fibrinogen adsorbed to the material surface compared to one with a flux of 13.0 ± 1.6 × 10(-10) mol NO cm(-2) min(-1) and the control (2334 ± 496, 226 ± 99, and 103 ±31% fibrinogen adsorbed of control, respectively). This study suggests that NO's role in controlling clotting is extended beyond platelet activation., (Copyright © 2013 Wiley Periodicals, Inc.)

Nitric oxide (NO) releasing (NORel) materials have been shown to create localized increases in NO concentration by the release of NO from a diazeniumdiolate-containing or S-nitrosothiol-containing polymer coating and the improvement of extracorporeal circulation (ECC) hemocompatibility. However, the mechanism and, in particular, the platelet upregulation of the NO/cGMP signaling protein, vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP (ser 239)), for the improved ECC hemocompatibility via NO release still needs elucidation. In this work, two NORel polymeric coatings were evaluated in a 4 h rabbit thrombogenicity model and the anti-thrombotic mechanism investigated for rabbit platelet P-VASP upregulation. Polymer films containing 25 wt% diazeniumdiolated dibutylhexanediamine (DBHD) or 5 wt% S-nitroso-N-acetylpenicillamine (SNAP) coated on the inner walls of ECC circuits yielded significantly reduced ECC thrombus formation and maintained normal platelet aggregation compared to polymer controls after 4 h of blood exposure. Platelet P-VASP (ser 239), a useful tool to monitor NO/cGMP signaling, was upregulated after 4 h on ECC and markedly increased after ex vivo sodium nitroprusside (SNP) stimulation. Interestingly, in the rabbit platelet, NO did not upregulate the cAMP P-VASP phosphoprotein P-VASP (ser 157) as previously shown in human platelets. These results suggest that NORel polymers preserve rabbit platelet quiescence by sustaining a level of cGMP signaling as monitored by P-VASP (ser 239) upregulation. The upregulation of this NO-mediated platelet signaling mechanism in this rabbit thrombogenicity model indicates the potential for improved thromboresistance of any NORel-coated medical device., (© 2013 Elsevier Ltd. All rights reserved.)

Tygon is a proprietary plasticized poly(vinyl chloride) polymer that is used widely in bioapplications, specifically as extracorporeal circuits. To overcome issues with blood clot formation and infection associated with the failure of these medical devices upon blood contact, we consider a Tygon coating with the ability to release the natural anticlotting and antibiotic agent, nitric oxide (NO), under simulated physiological conditions. These coatings are prepared by incorporating 20 w/w% S-nitrosoglutathione (GSNO) donor into a Tygon matrix. These films release NO on the order of 0.64 ± 0.5 × 10(-10) mol NO cm(-2) min(-1), which mimics the lower end of natural endothelium NO flux. We use a combination of assays to quantify the amount of GSNO that is found intact at different time points throughout the film soak, as well as monitor the total thiol content in the soaking solution due to any analyte that has leached from the polymer film. We find that a burst of GSNO is released from the material surface within 5 min to 1 h of soaking, which only represents 0.25% of the total GSNO contained in the film. After 1 h of film soak, no additional GSNO is detected in the soaking solution. By further considering the total thiol content in solution relative to the intact GSNO, we demonstrate that the amount of GSNO leached from the material into the buffer soaking solution does not contribute significantly to the total NO released from the GSNO-incorporated Tygon film (<10% total NO). Further surface analysis using SEM-EDS traces the elemental S on the material surface, demonstrating that within 5 min -1 h soaking time, 90% of the surface S is removed from the material. Surface wettability and roughness measurements indicate no changes between the GSNO-incorporated films pre- to postsoak that will be significant toward the adsorption of biological components, such as proteins, relative to the presoaked donor-incorporated film. Overall, we demonstrate that, for a 20 w/w% GSNO-incorporated Tygon film, relatively minimal GSNO leaching is experienced, and the lost GSNO is from the material surface. Varying the donor concentration from 5 to 30 w/w% GSNO within the film does not result in significantly different NO release profiles. Additionally, the steady NO flux associated with the system is predominantly due to localized release from the material, and not donor lost to soaking solution. The surface properties of these materials generally imply that they are useful for blood-contacting applications.

Nitric oxide (NO) inhibits platelet aggregation at and near the site of a vascular injury by upregulation of cyclic guanosine monophosphate, which reduces the dimerization of the integrin α(IIb)β₃. The magnitude of NO flux from the vessel wall and the NO concentration that is necessary to inhibit platelet aggregation under physiological flow conditions is unknown. In this study, a NO releasing polymer, diazeniumdiolated dibutylhexanediamine, was integrated into a microfluidic flow assay to determine the relationship between NO wall flux and collagen mediated platelet adhesion, activation and aggregation. A NO flux equal to or greater than 2.5 × 10⁻¹⁰ mol cm⁻² min⁻¹ was found to abrogate aggregation, but not initial platelet adhesion, on collagen at 200 and 500 s⁻¹ as effectively as the α(IIb)β₃ antagonist abciximab. The dynamic range of NO fluxes found to induce measurable inhibition of platelet aggregation spanned from 0.33 × 10⁻¹⁰ to 2.5 × 10⁻¹⁰ mol cm⁻² min⁻¹ at 200-500 s⁻¹. These fluxes correspond to near-wall NO concentrations of 3-90 nM based on a computational model of NO transport. The model predicts that NO concentration in the platelet rich layer near the wall is kinetically limited, while NO penetration into the lumen is mass transfer limited.

Despite the popularity of walking as a form of physical activity for obese individuals, relatively little is known about how obesity affects the metabolic rate, economy, and underlying mechanical energetics of walking across a range of speeds and grades. The purpose of this study was to quantify metabolic rate, stride kinematics, and external mechanical work during level and gradient walking in obese and nonobese adults. Thirty-two obese [18 women, mass = 102.1 (15.6) kg, BMI = 33.9 (3.6) kg/m(2); mean (SD)] and 19 nonobese [10 women, mass = 64.4 (10.6) kg, BMI = 21.6 (2.0) kg/m(2)] volunteers participated in this study. We measured oxygen consumption, ground reaction forces, and lower extremity kinematics while subjects walked on a dual-belt force-measuring treadmill at 11 speeds/grades (0.50-1.75 m/s, -3° to +9°). We calculated metabolic rate, stride kinematics, and external work. Net metabolic rate (Ė net/kg, W/kg) increased with speed or grade across all individuals. Surprisingly and in contrast with previous studies, Ė net/kg was 0-6% less in obese compared with nonobese adults (P = 0.013). External work, although a primary determinant of Ė net/kg, was not affected by obesity across the range of speeds/grades used in this study. We also developed new prediction equations to estimate oxygen consumption and Ė net/kg and found that Ė net/kg was positively related to relative leg mass and step width and negatively related to double support duration. These results suggest that obesity does not impair walking economy across a range of walking speeds and grades.

Tumor resistance to cytotoxic therapeutics coupled with dose-limiting toxicity is a serious hurdle in the field of medical oncology. In the face of this obstacle, nitric oxide has emerged as a powerful adjuvant for the hypersensitization of tumors to more traditional chemo- and radio-therapeutics. Furthermore, emerging evidence indicates that nitric oxide donors have the potential to function independently in the clinical management of cancer. Herein, we discuss the role of nitric oxide in cancer and the potential for nitric oxide donors to support conventional therapeutics., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Many common wound healing aids are created from biodegradable polymeric materials. Often, these materials are unable to induce complete healing in wounds because of their failure to prevent infection and promote cell growth. This study reports the development of therapeutic materials aimed at overcoming these limitations through the release of a naturally occurring antimicrobial agent from a porous, polymeric fiber scaffold. The antimicrobial character was achieved through the release of nitric oxide (NO) while the porous structure was fabricated through electrospinning polymers into nanofibers. Three variations of the polymer poly(lactic-co-glycolic-co-hydroxymethyl propionic acid) (PLGH) modified to include thiol and NO groups were investigated. Fibers of the modified polymers exhibited smooth, bead free morphologies with diameters averaging between 200 and 410 nm. These fibers were deposited in a random manner to create a highly porous fibrous scaffold. The fibers were found to release NO under physiological pH and temperature and have the capacity to release 0.026 to 0.280 mmol NO g(-1). The materials maintained their fibrous morphological structure after this exposure to aqueous conditions. The sustained morphological stability of the fiber structure coupled to their extended NO release gives these materials great potential for use in wound healing materials.

This article draws on theories of gender inequality and immigrant health to hypothesize differences among the largest immigrant population, Mexicans, and a lesser known population of Middle Easterners. Using data from the 2000-2007 National Health Interview Surveys, we compare health outcomes among immigrants to those among U.S.-born whites and assess gender differences within each group. We find an immigrant story and a gender story. Mexican and Middle Eastern immigrants are healthier than U.S.-born whites, and men report better health than women regardless of nativity or ethnicity. We identify utilization of health care as a primary mechanism that contributes to both patterns. Immigrants are less likely than U.S.-born whites to interact with the health care system, and women are more likely to do so than men. Thus, immigrant and gender health disparities may partly reflect knowledge of health status rather than actual health.

The use of metal organic frameworks (MOFs) for the catalytic production of nitric oxide (NO) is reported. In this account we demonstrate the use of Cu(3)(BTC)(2) as a catalyst for the generation of NO from the biologically occurring substrate, S-nitrosocysteine (CysNO). The MOF catalyst was evaluated as an NO generator by monitoring the evolution of NO in real time via chemiluminescence. The addition of 2, 10, and 15-fold excess CysNO to MOF-Cu(II) sites and cysteine (CysH) resulted in catalytic turnover of the active sites and nearly 100% theoretical yield of the NO product. Control experiments without the MOF present did not yield appreciable NO generation. In separate studies the MOF was found to be reusable over successive iterations of CysNO additions without loss of activity. Subsequently, the MOF catalyst was confirmed to remain structurally intact by pXRD and ATR-IR following reaction with CysNO and CysH.

An understanding of the nitrosation processes that dictate S-nitrosothiol formation in the presence of a polymer is crucial toward the controlled synthesis of nitric oxide (NO)-releasing materials, an important class of biomaterials that mimic the natural function of cells. Herein, the kinetics of S-nitrosoglutathione (GSNO) formation in the presence of dextran under a variety of nitrosation conditions, including the nitrosating agent and the dextran concentration, are reported. When comparing nitrous acid and t-butyl nitrite as the nitrosating agent, the use of nitrous acid results in 100% nitrosation of the thiol sites within less than a minute and t-butyl nitrite requires more than 5 min to reach completion. This trend establishes nitrous acid as a highly efficient nitrosating agent. In the presence of increasing dextran concentration from 0 w/v% to 10 w/v%, the extent of nitrosation decreases by approximately 5% and 30% using nitrous acid and t-butyl nitrite, respectively. With sufficient reaction time, either reagent leads to 100% nitrosation. This indicates that t-butyl nitrite is the preferred reagent for fine-tuned NO loading of thiol sites as the extent of reaction is greatly impacted by the polymer concentration. Taken together, these studies provide valuable insights regarding the ability to tailor NO storage within biomaterials for use in a wide range of clinical applications.

TatC (STM3975) is a highly conserved component of the Twin Arginine Transport (Tat) systems that is required for transport of folded proteins across the inner membrane in gram-negative bacteria. We previously identified a ΔtatC mutant as defective in competitive infections with wild type ATCC14028 during systemic infection of Salmonella-susceptible BALB/c mice. Here we confirm these results and show that the ΔtatC mutant is internalized poorly by cultured J774-A.1 mouse macrophages a phenotype that may be related to the systemic infection defect. This mutant is also defective for short-term intestinal and systemic colonization after oral infection of BALB/c mice and is shed in reduced numbers in feces from orally infected Salmonella-resistant (CBA/J) mice. We show that the ΔtatC mutant is highly sensitive to bile acids perhaps resulting in the defect in intestinal infection that we observe. Finally, the ΔtatC mutant has an unusual combination of motility phenotypes in Salmonella; it is severely defective for swimming motility but is able to swarm well. The ΔtatC mutant has a lower amount of flagellin on the bacterial surface during swimming motility but normal levels under swarming conditions.

As the world of critical care medicine advances, extracorporeal therapies (ECC) have become commonplace in the management of the high risk intensive care patient. ECC encompasses a wide variety of technologies from hemodialysis, continuous renal replacement therapy (CRRT) and plasmapheresis, to cardiopulmonary bypass (CPB), extracorporeal life support (ECLS) and hepatic support. The development of internal man made organs is the next step with ventricular assist devices and artificial lungs. As we advance the technologies with smaller devices, and more intricate circuitry, we lack the keystone necessary to control the blood-biomaterial interface. For the last 50 years much has been learned about surface induced thrombosis and attempts have been made to prevent it with alternative systemic anticoagulation, circuitry surface modifications, or a combination of both. Despite these efforts, systemic or regional anticoagulation remain necessary for both laboratory and clinical application of ECC. As such, the development of an endothelial-like, biomimetic surface to reduce or perhaps even eliminate the blood activation/thrombus formation events that occur upon exposure to artificial surfaces is paramount.

Nitric oxide (NO) has been shown to reduce thrombogenicity by decreasing platelet and monocyte activation by the surface glycoprotein, P-selectin and the integrin, CD11b, respectively. In order to prevent platelet and monocyte activation with exposure to an extracorporeal circulation (ECC), a nitric oxide releasing (NORel) polymeric coating composed of plasticized polyvinyl chloride (PVC) blended with a lipophilic N-diazeniumdiolate was evaluated in a 4 h rabbit thrombogenicity model using flow cytometry. The NORel polymer significantly reduced ECC thrombus formation compared to polymer control after 4 h blood exposure (2.8 +/- 0.7 NORel vs 6.7 +/- 0.4 pixels/cm(2) control). Platelet count (3.4 +/- 0.3 NORel vs 2.3 +/- 0.3 x 10(8)/ml control) and function as measured by aggregometry (71 +/- 3 NORel vs 17 +/- 6% control) were preserved after 4 h exposure in NORel versus control ECC. Plasma fibrinogen levels significantly decreased in both NORel and control groups. Platelet P-selectin mean fluorescence intensity (MFI) as measured by flow cytometry was attenuated after 4 h on ECC to ex vivo collagen stimulation (27 +/- 1 NORel vs 40 +/- 2 MFI control). Monocyte CD11b expression was reduced after 4 h on ECC with NORel polymer (87 +/- 14 NORel vs 162 +/- 30 MFI control). These results suggest that the NORel polymer coatings attenuate the increase in both platelet P-selectin and monocytic CD11b integrin expression in blood exposure to ECCs. These NO-mediated platelet and monocytic changes were shown to improve thromboresistance of these NORel-polymer-coated ECCs for biomedical devices., (2009 Elsevier Ltd. All rights reserved.)

Nitric oxide (NO) has been shown to exhibit significant anti-platelet activity and its release from polymer matrices has been already utilized to increase the biocompatibility of various blood-contacting devices. Herein, details of a new synthetic approach for preparing NO-releasing diazeniumdiolated polyurethanes (PU) are described. The method's utility is demonstrated by the incorporation of methoxymethyl- or sugar-protected pre-formed diazeniumdiolate moieties directly into chain extender diols which are then incorporated into the polyurethane backbone. This approach provides the ability to control the number of diazeniumdiolate groups incorporated into the polymer backbone, and hence the surface flux of NO that can ultimately be liberated from polymeric films prepared from the new PU materials. The method provides a means of covalently attaching diazeniumdiolate groups to polyurethanes in a form that resists dissociation of NO during processing but can be activated for spontaneous NO release via hydrolysis of the carbohydrate or methoxymethyl moieties under basic and acidic conditions, respectively.

Polyurethanes are widely used in the manufacturing of biomedical catheters and other blood-contacting devices; however, thrombus formation still occurs, which renders these catheters ineffective unless systemic anticlotting agents are used. Nitric oxide (NO) is a well-known inhibitor of platelet activity. In the current study, two commercially available medical polyurethanes (Pellethane and Tecophilic) were derivatized to possess NO-generating Cu(II)-cyclen moieties pendant to the polymer backbone. A new three-step synthetic approach is used, that is simpler than a recently reported method to prepare Cu(II)-cyclen-polyurethane materials. Both derivatized polyurethanes were found to produce NO at levels at or above those of endothelial cells. A comparison between the modified commercial polyurethanes (hydrophobic vs. hydrophilic) is presented, including the synthetic scheme, extensive characterization, and coating application. These derivatized polymers may serve as useful coatings to prevent clotting on the surface of catheters and other blood-contacting biomedical devices., ((c) 2009 Wiley Periodicals, Inc.)

Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G(2)/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G(1). These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G(1) induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS).

The title compound, C(10)H(24)N(6)O(4), is the most stable type of nitric oxide (NO) donor among the broad category of discrete N-diazeniumdiolates (NO adducts of nucleophilic small molecule amines). Sitting astride a crystallographic inversion center, the molecule contains a symmetric dimethylhexane-1,6-diamine structure bearing two planar O(2)-methylated N-diazeniumdiolate functional groups [N(O)=NOMe]. These two groups are parallel to each other and have the potential to release four molecules of NO. The methylated diazeniumdiolate substituent removes the negative charge from the typical N(O)=NO(-) group, thereby increasing the stability of the diazeniumdiolate structure. The crystal was nonmerohedrally twinned by a 180 degrees rotation about the real [101] axis. This is the first N-based bis-diazeniumdiolate compound with a flexible aliphatic main unit to have its structure analyzed and this work demonstrates the utility of stabilizing the N-diazeniumdiolate functional group by methylation.

The 2-hydroxy-5-nitrobenzyl group is shown to be an effective protecting group for diazeniumdiolates. O(2)-(2-hydroxy-5-nitrobenzyl)-substituted diazeniumdiolates display enhanced thermal stability, but efficiently release nitric oxide (NO) in pH 7.4 aqueous solutions. A lipophilic analogue incorporated into hydrophobic polymers shows NO surface flux rates comparable to that of the natural endothelium. Importantly, these polymer formulations also show significantly enhanced biocompatibility in vivo with use of a porcine implant model.

The gold standard for anticoagulation during extracorporeal circulation (ECC) remains systemic heparinization and the concomitant risk of bleeding in an already critically ill patient could lead to death. Normal endothelium is a unique surface that prevents thrombosis by the release of antiplatelet and antithrombin agents. Nitric oxide (NO) is one of the most potent, reversible antiplatelet agents released from the endothelium. Nitric oxide released from within a polymer matrix has been proven effective for preventing platelet activation and adhesion onto extracorporeal circuits. However, the critical NO release (NO flux) threshold for thrombus prevention during ECC has not yet been determined. Using a 4-hour arteriovenous (AV) rabbit model of ECC, we sought to find this threshold value for ECC circuits, using an improved NO-releasing coating (Norel-b). Four groups of animals were tested at variable NO flux levels. Hourly blood samples were obtained for measurement of arterial blood gases, platelet counts, fibrinogen levels and platelet function (via aggregometry). A custom-built AV circuit was constructed with 36 cm of poly(vinyl)chloride (PVC) tubing, a 14 gauge (GA) angiocatheter for arterial access and a modified 10 French (Fr) thoracic catheter for venous access. The Norel-b coating reduced platelet activation and thrombus formation, and preserved platelet function - in all circuits that exhibited an NO flux of 13.65 x 10(10) mol x cm(-2) x min(-1). These results were significant when compared with the controls. With the Norel-b coating, the NO flux from the extracorporeal circuit surface can be precisely controlled by the composition of the polymer coating used, and such coatings are shown to prevent platelet consumption and thrombus formation while preserving platelet function in the animal.

Two novel strategies for synthesizing stable polyurethanes (PUs) capable of generating bioactive nitric oxide (NO) are described. The methods rely on covalently attaching diazeniumdiolate (N(2)O(2)(-)) groups onto secondary amine nitrogens at various positions within the polymer chain such that, when in contact with water or physiological fluids, only the two molecules of NO available from each diazeniumdiolate moiety are released into the surrounding medium, with potential byproducts remaining covalently bound to the matrix. Extensive analysis of the NO(x)() products released from the polymers was employed to develop appropriate strategies to better stabilize the diazeniumdiolate-based polymer structures. In one approach, diazeniumdiolate groups are attached to secondary amino nitrogens of alkane diamines inserted within the diol chain extender of a PU material. Oxidative loss of NO was minimized by blending the polymer with a biocompatible, relatively nonnucleophilic salt before exposing solutions of the polymer to NO during the diazeniumdiolation step. Fluxes of molecular NO from such materials during immersion in physiological buffer reached levels as high as 19 pmol x cm(-2) x s(-1) with a total recovery of 21 nmol of NO/mg of PU. A second general synthetic strategy involved omega-haloalkylating the urethane nitrogens and then displacing the halide from the resulting polymer with a nucleophilic polyamine to form a PU with pendent amino groups suitable for diazeniumdiolation. Commercially available Pellethane 2363-80AE that was bromobutylated and then reacted with diethylenetriamine and further exposed to gaseous NO proved stable in solid form for several months, but released NO with a total recovery of 17 nmol/mg upon immersion in physiological buffer. This material showed an initial NO flux of 14 pmol x cm(-2) x s(-1) when immersed in pH 7.4 buffer at 37 degrees C, with gradually decreasing but still observable fluxes for up to 6 days.

Diazeniumdiolate ions [R2N-N(O)=N-O-] are of growing interest pharmacologically for their ability to generate up to two molar equivalents of bioactive nitric oxide (NO) spontaneously on protonating the amino nitrogen. Accordingly, their stability increases as the pH is raised. Here we show that the corresponding beta-glucosides [R2N-N(O)=N-O-Glc] decreased in stability with pH; when R2N was diethylamino, the rate equation was kobs = ko + kOH- [OH-], where ko = 7.8 x 10-7 s-1 and kOH- = 5.3 x 10-3 M-1 s-1. The primary products were 1,6-anhydroglucose and the regenerated R2N-N(O)=N-O- ion. The results were qualitatively similar to those of beta-glucosyl fluoride and p-nitrophenoxide, whose hydrolyses have been rationalized as proceeding via a glycal oxide intermediate. This chemistry offers a convenient strategy for protecting heat- and acid-sensitive diazeniumdiolate ions during manipulations that would otherwise destroy them. As an example, a poly(urethane) film that generated NO in physiological buffer at a surface flux comparable to that of the mammalian vascular endothelium was prepared by glucosylating the ionic diazeniumdiolate group attached to the diol monomer before reacting it with the bis-isocyanate, then removing the saccharide with base when the protecting group was no longer needed.

[reaction: see text] The synthesis and characterization of a series of symmetric bis-dialkyldiamine-based diazeniumdiolates, RN[N(O)NO(-)Na(+)](CH(2))(x)()N[N(O)NO(-)Na(+)]R', are reported. Preparation of corresponding intramolecular diazeniumdiolates of the form RN[N(O)NO](-)(CH(2))(x)()NH(2)(+)R' with alkyl groups > (CH(2))(4)CH(3) have been shown previously to lack stability. In contrast, sodium-stabilized bis-diazeniumdiolates of such lipophilic species can be readily formed when these same diamines are reacted with NO in basic media. The resulting compounds release 4 mol of NO per mole of original diamine. This approach enables the synthesis of more lipophilic NO donors than previously possible.

The current state-of-the-art with respect to the preparation, characterization and biomedical applications of novel nitric oxide (NO) releasing or generating polymeric materials is reviewed. Such materials show exceptional promise as coatings to prepare a new generation of medical devices with superior biocompatiblity. Nitric oxide is a well-known inhibitor of platelet adhesion and activation, as well as a potent inhibitor of smooth muscle cell proliferation. Hence, polymers that release or generate NO locally at their surface exhibit greatly enhanced thromboresistivity and have the potential to reduce neointimal hyperplasia caused by device damage to blood vessel walls. In this review, the use of diazeniumdiolates and nitrosothiols as NO donors within a variety polymeric matrixes are summarized. Such species can either be doped as discrete NO donors within polymeric films, or covalently linked to polymer backbones and/or inorganic polymeric filler particles that are often employed to enhance the strength of biomedical polymers (e.g., fumed silica or titanium dioxide). In addition, very recent efforts to create catalytic polymers possessing immobilized Cu(II) sites capable of generating NO from endogenous oxidized forms of NO already present in blood and other physiological fluids (nitrite and nitrosothiols) are discussed. Preliminary literature data illustrating the efficacy of the various NO release/generating polymers as coatings for intravascular sensors, extracorporeal blood loop circuits, and arteriovenous grafts/shunts are reviewed.

Objectives: Nitric oxide (NO), produced by normal vascular endothelial cells, reduces platelet aggregation and thrombus formation. NO-releasing biopolymers have the potential to prolong vascular graft and stent patency without adverse systemic vasodilation., Methods: 5-mm polyurethane vascular grafts coated with a polymer containing the NO-donor dialkylhexanediamine diazeniumdiolate were implanted for 21 days in a sheep arteriovenous bridge-graft model., Results: Eighty percent (4/5) of grafts coated with the NO-releasing polymer remained patent through the 21 day implantation period, compared to fifty percent (2/4) of sham-coated grafts and no (0/3) uncoated grafts. Thrombus-free surface area (+/-SEM) of explanted grafts was significantly increased in NO-donor coated grafts (98.2% +/- 0.9%) compared with sham-coated (79.2% +/- 8.6%) and uncoated (47.2% +/- 5.4%) grafts ( P = .00046). Examination of the graft surface showed no adherent thrombus or platelets and no inflammatory cell infiltration in NO-donor coated grafts, while control grafts showed adherent complex surface thrombus consisting of red blood cells in an amorphous fibrin matrix, as well as significant red blood cell and inflammatory cell infiltration into the graft wall., Conclusion: In this study we determined that local NO release from the luminal surface of prosthetic vascular grafts can reduce thrombus formation and prolong patency in a model of prosthetic arteriovenous bridge grafts in adult sheep. These findings may translate into improved function and improved primary patency rates in small-diameter prosthetic vascular grafts.

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.

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.

In pines, self-pollination rates can be as high as 34% yet only 5% of viable seed is a product of self-fertilization. This decline in selfed seed viability is the consequence of post-fertilization exclusion mechanisms operating via the embryo lethal system. Recent molecular marker dissection studies suggest that the embryo lethal system is composed of semilethal factors dispersed across the genome, but it is not clear whether overdominant lethal factors are rare or representative. The study objective was to determine if overdominance was rare for the embryo lethal system in conifers. Three cohorts of selfed offspring from a single Pinus taeda parent were genotyped for nuclear microsatellites. Maximum likelihood tests based on distorted segregation ratios for single markers and for interval mapping were used to infer the degree of dominance. Four hypotheses about overdominance lethal factors were tested: (1) overdominant lethal factors rarely occur within the embryo lethal system, (2) overdominant lethal factors are rarely detected because they are transient and display stage-specific expression, (3) overdominant lethal factors are rarely detected due to tight linkage with rare marker alleles and (4) dominance estimation is unbiased by gametic selection. Four out of the seven chromosomal segments were linked to an overdominant lethal factor. One of these four segments had symmetric overdominance, an effect which persisted from embryo maturity through germination. Four overdominant lethal factors were linked to common and rare marker alleles. Gametic selection was not a source of bias in dominance estimation. Overdominant or pseudo-overdominant lethal factors are a common component of the conifer embryo lethal system.

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.

A consensus conference on the use of placebo in mood disorder studies consisted of expert presentations on bioethics, biostatistics, unipolar depression, and bipolar disorder. Work groups considered evidence and presented statements to the group. Although it was not possible to write a document for which there was complete agreement on all issues, the final document incorporated input from all authors. There was consensus that placebo has a definite role in mood disorder studies. Findings of equivalence between a new drug and standard treatment in active control studies is not evidence of efficacy unless the new drug is also significantly more effective than placebo. Add-on studies in which patients are randomized to standard therapy plus the investigational drug or standard therapy plus placebo are especially indicated for high-risk patients. Mood disorders in elderly and pediatric patients are understudied, and properly designed trials are urgently needed. Research is needed on the ethical conduct of studies to limit risks of medication-free intervals and facilitate poststudy treatment. Patients must fully understand the risks and lack of individualized treatment involved in research.

The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival.

The increasing importance of the acquired immunodeficiency syndrome (AIDS) as a cause of large, clinically significant pericardial effusions has not been well documented. To determine the frequency and characteristics of large AIDS-associated pericardial effusions, we reviewed the records of 50 consecutive patients undergoing pericardiocentesis between 1985 and 1990; AIDS was the most common underlying illness and was present in 14 patients (28 percent). The pericardial fluid was diagnostic in three (21 percent) of the 14 cases (one bacterial, one positive for acid-fast bacilli, and one lymphoma). Of the 11 patients with nondiagnostic fluid, one underwent a pericardial biopsy which revealed granuloma consistent with mycobacterial disease, four had active pulmonary tuberculosis (TB), and two responded clinically to anti-TB therapy. Thus, in 8 (57 percent) of the 14 patients with AIDS, there was either definitive or suggestive evidence of mycobacterial disease. We conclude that AIDS is now a common underlying illness associated with large pericardial effusions and that mycobacterial disease may frequently be the etiology.

We determined whether normal human lung fibroblasts expressed cell-associated thymocyte-stimulating activity in response to recombinant interleukin-1 (rIL-1) (alpha and beta) and recombinant tumor necrosis factor (rTNF). Individually, rIL-1 and rTNF induced fibroblast expression of thymocyte-stimulating activity, with rIL-1 being significantly more potent. Importantly, combining rIL-1 and rTNF resulted in a synergistic increase in fibroblast thymocyte-stimulating activity. This synergistic interaction was dose dependent for both cytokines and was not noted when gamma-interferon was combined with rIL-1 or rTNF. In all cases, the thymocyte-stimulating activity was the result of an IL-1 alpha-like moiety whose maximal production required protein synthesis. IL-1 alpha activity could be detected after as little as 4 h, peaked after 24 h, and returned toward normal with longer periods of cytokine-fibroblast incubation. However, cytokine-stimulated fibroblasts that no longer expressed IL-1 alpha activity could be induced to re-express this activity with repeat cytokine challenge. Induction of fibroblast IL-1 alpha by IL-1 and/or TNF may be an important mechanism amplifying IL-1-mediated biologic events at sites of local inflammation.

It is estimated that fifteen percent of the population is in need of some kind of mental health service at any given time, thus constituting a primary health problem. The President's Commission on Mental Health (PCMH) recognized that religious institutions can help to prevent mental illness by providing support in the community. This paper presents types of programs the PCMH found that were supportive and describes the program of one church to illustrate additional ways that clergy and their congregants, working collaboratively with professionals and agencies, can contribute significantly to the prevention of mental illness.

The question of professional accountability has frequently been raised with regard to the provision of health care services. Various systems, among them peer review, utilization review, and the PSRO, have accordingly been introduced to monitor the quality and the cost of health care. Social workers will have increased opportunities to participate in these review systems and, thus, to improve health care delivery.

Although the number of social workers participating in medical education is increasing, there is a need to review and evaluate their roles in such educational activities. Information is needed about how social workers participate in medical education in order to plan how to make a more effective contribution. This article describes how the role of social workers in medical education evolved, and gives examples of what social workers have done in the past and are doing today.

To understand the role fibroblasts play in mediating and amplifying the effects of inflammatory cytokines, we determined whether recombinant interleukin 1 (IL-1) and recombinant tumor necrosis factor (TNF), alone and in combination, stimulated fibroblasts to produce IL-1 beta. Recombinant IL-1 (alpha and beta) stimulated fibroblast IL-1 beta mRNA accumulation, whereas recombinant TNF did not. In addition, simultaneous stimulation with recombinant IL-1 (alpha or beta) and recombinant TNF resulted in a synergistic increase in IL-1 beta mRNA levels. However, in all cases, IL-1 beta mRNA accumulation was not associated with fibroblast production of soluble IL-1 beta protein. Lysates of unstimulated, recombinant IL-1-stimulated, and recombinant TNF-stimulated fibroblasts did not contain IL-1 beta prohormone. In contrast, IL-1 beta prohormone was detected in lysates of fibroblasts incubated simultaneously with recombinant IL-1 and recombinant TNF. These studies demonstrate that recombinant IL-1 stimulates fibroblast IL-1 beta mRNA accumulation and that recombinant IL-1 and recombinant TNF synergize to further up-regulate IL-1 beta mRNA levels. In addition, they show that IL-1 beta production by human lung fibroblasts is inhibited at a posttranscriptional level. Translational control appears to be important in recombinant IL-1-stimulated fibroblasts and posttranslational control is important in fibroblasts stimulated simultaneously with recombinant IL-1 and recombinant TNF.