Modeling Water Quality in Watersheds: From Here to the Next Generation.

In this synthesis, we assess present research and anticipate future development needs in modeling water quality in watersheds. We first discuss areas of potential improvement in the representation of freshwater systems pertaining to water quality, including representation of environmental interfaces, in-stream water quality and process interactions, soil health and land management, and (peri-)urban areas. In addition, we provide insights into the contemporary challenges in the practices of watershed water quality modeling, including quality control of monitoring data, model parameterization and calibration, uncertainty management, scale mismatches, and provisioning of modeling tools. Finally, we make three recommendations to provide a path forward for improving watershed water quality modeling science, infrastructure, and practices. These include building stronger collaborations between experimentalists and modelers, bridging gaps between modelers and stakeholders, and cultivating and applying procedural knowledge to better govern and support water quality modeling processes within organizations.

Wound-Induced Proteinase Inhibitor in Plant Leaves: A Possible Defense Mechanism against Insects.

Wounding of the leaves of potato or tomato plants by adult Colorado potato beetles, or their larvae, induces a rapid accumulation of a proteinase inhibitor throughout the plants' tissues that are exposed to air. This effect of insect damage can be simulated by mechanically wounding the leaves. The transport of a factor out of damaged leaves takes place rapidly after the wound is inflicted and the levels of proteinase inhibitor, in both damaged and adjacent leaves, rises strikingly within a few hours. The rapid accumulation of a powerful inhibitor of major intestinal proteinases of animals in response to wounding of the leaves is probably a defense mechanism.

Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils.

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.

Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils.

The subcellular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotaurine and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50:1. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiring cells by exposure to opsonized zymosan. These results prompted in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypotaurine competes with 5,5'-dimethyl-1-pyrroline N-oxide) (DMPO) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.

Beta-carotene in HIV infection: an extended evaluation.

OBJECTIVE: Several small short-term intervention studies have suggested that beta-carotene supplementation in HIV-infected patients can increase the number of various immune cells including CD4 cells. This prospective double-blinded study was designed to investigate whether beta-carotene supplementation would result in this immuno-enhancement in a larger number of patients over a longer time period. METHODS: HIV-positive patients were randomly assigned to receive either 60 mg beta-carotene orally three times daily or a matched placebo. In addition, all patients received a multivitamin supplement. Patients were evaluated at baseline, 1 month, and 3 months for T-cell quantitative subsets, natural killer cells, HIV p24 antigen, beta-carotene levels, complete blood counts and chemistry batteries. Body weights and Karnofsky scores were evaluated at each visit. RESULTS: Seventy-two patients signed informed consent forms and entered the study. Except for serum beta-carotene concentration, there were no statistically significant differences (P < 0.05) between the treatment (60 mg beta-carotene three times daily and multivitamins) and placebo (placebo and multivitamins) groups at baseline or after either 1 or 3 months of treatment. DISCUSSION: Earlier studies suggesting that beta-carotene supplementation increased levels of immune cells in HIV-infected patients were not replicated in this study. The addition of a multivitamin supplement to both arms of this study may have masked any difference between the two groups. However, on the basis of the results of this study, we would not recommend supplementation with high doses of beta-carotene for HIV-infected patients.

Detection of NADPH diaphorase activity associated with human neutrophil NADPH-O2 oxidoreductase activity.

At approximately equimolar concentrations (approximately 70 microM), and in the presence of excess catalase and superoxide dismutase, DCIP, ferricytochrome c and ferricyanide abstracted 21, 6 and 61%, respectively, of the electron equivalents given up by NADPH to the NADPH-O2 oxidoreductase complex derived from phorbol myristate acetate-stimulated human neutrophils. With a 10-fold increase in ferricyanide, all of the electron equivalents given up by NADPH to the oxidoreductase complex were shunted to ferricyanide concomitant with complete inhibition of NADPH-dependent O2 consumption. These results substantiate the existence of intrinsic diaphorase activity associated with the superoxide generating NADPH-O2 oxidoreductase of human neutrophils.

Myeloperoxidase oxidation of sulfur-centered and benzoic acid hydroxyl radical scavengers.

Myeloperoxidase (MPO) oxidizes sulfur-centered and benzoate hydroxyl radical scavengers through formation of HOCl. Sulfur-centered hydroxyl radical scavengers compete with benzoate as antioxidants of HOCl. We conclude from these observations that competition experiments between benzoate and sulfur-centered hydroxyl radical scavengers are not sufficiently specific to infer participation of hydroxyl radicals in oxidative reactions mediated by neutrophils because of the unique action of MPO in affecting oxidation of the test radical scavengers.

Production of TNF-alpha and bone resorbing activity by macrophages in response to different types of bone cement particles.

We have compared the capacity of clinically relevant wear debris from seven different cement types to activate macrophages to produce TNF-alpha, IL-1beta, IL-6 and bone resorbing activity in vitro. The bone cements were: CMW 1 original (PMMA only); CMW 1RO (1 microm BaSO4; 9.2%); CMW copolymer bone cement 1 (10 microm BaSO4; 10%); CMW copolymer bone cement 2 (1 microm BaSO4; 10%); Palacos R (10 microm ZrO2; 15.6%); CMW Calcium phosphate cement 20% (10 microm tri-calcium phosphate; 20%) and CMW calcium phosphate cement 30% (10 microm tri-calcium phosphate; 30%). Cement debris was produced aseptically using a simple configuration wear test. The majority of particles were in the size range 0.1-0.5 microm for each cement type. The cement particles were co-cultured with the U937 macrophage cell line at ratios of 10 and 100 microm3 particle volumes to macrophage cell numbers for 24 h. At the 10:1 ratio the particles had no effect on the cells. At the 100:1 ratio, the major cytokine produced was TNF-alpha and there were no statistical differences between the different types of cement debris. The bone resorption activity of the co-culture supernatants was significantly greater than the control (U937 cells without particles) for particles of CMW 1RO, CMW copolymer bone cement 1, CMW copolymer bone cement 2 and Palacos R (P < 0.05, ANOVA). However there were no statistical differences between the levels of bone resoprtion evoked by these four cement types. The CMW1 original and CMW calcium phosphate containing cements failed to induce the macrophages to elaborate bone resorption activity at the 100:1 ratio. These data suggest that the addition of radio-opaque additives to bone cement may increase the capacity of the debris to induce osteolysis.

Comparison of the response of primary human peripheral blood mononuclear phagocytes from different donors to challenge with model polyethylene particles of known size and dose.

The response of primary human peripheral blood mononuclear phagocytes to challenge with polyethylene particles of known size and dose was evaluated. Particles with mean sizes of 0.21, 0.49, 4.3, 7.2, and 88 microm were co-cultured with cells for 24 h prior to the assessment of cell viability and production of the osteolytic mediators IL-1beta, IL-6, TNFalpha, GM-CSF and PGE2. All particle fractions were evaluated at particle volume (microm3) to cell number ratios of 10:1 and 100:1 which were previously identified as being the most biologically active and clinically relevant. The heterogeneity of human individuals was clearly evident both in the profile and the magnitude of the response of the donors evaluated in this study (the response of donor 5 being 2- to 15-fold lower than that of the other donors). Only the sub-micrometre particles stimulated significantly enhanced cytokine secretion at the ratios tested: mean particle sizes of 0.49 and 0.21 microm being the most biologically active. Macrophages stimulated with particles outside this size range produced considerably lower levels of mediator. These results compared favourably with the results of earlier studies, which demonstrated that particles within the phagocytosable size range (0.1-10 microm) were the most biologically active. These results, therefore, confirm earlier findings and suggest that the size and volume of polyethylene particles are critical factors in macrophage activation. Furthermore, they suggest that the heterogeneity of human individuals may be another important factor in determining implant life and could provide the basis for a valuable diagnostic tool to identify those patients most at risk of implant loosening.

Polyethylene particles of a 'critical size' are necessary for the induction of cytokines by macrophages in vitro.

Particulate wear debris from total hip prosthetic components can stimulate macrophages to produce mediators of osteolysis which may cause aseptic implant loosening. This study evaluated the in vitro response of murine peritoneal macrophages to polyethylene particles of definitive size distributions at varying volume doses. Ceridust 3615 polyethylene particles with a mean size of 0.21, 0.49, 4.3 and 7.2 microm and GUR 120 polyethylene resin with a mean size of 88 microm were co-cultured with C3H murine peritoneal macrophages at volume (microm)3 to cell number ratios of 100:1, 10:1, 1:1 and 0.1: 1. The secretion of IL-6, IL-1beta and TNF-alpha was determined by ELISA. Significantly elevated levels of TNF-alpha and IL-1beta were determined at 100:1 ratios when the macrophages were challenged with particles with a mean size of 0.49, 4.3 and 7.2 microm, and at 10:1 ratios for particles with a mean size of 0.49 and 4.3 microm. IL-6 production was significantly elevated at 100:1 ratios for mean particle sizes of 0.49 and 4.3 microm. Particles outside this range produced considerably less cytokine suggesting that both the size and volume (or number) of polyethylene particles are critical factors in macrophage activation. Therefore particles in the phagocytosable size range of 0.3-10 microm appear to be the most biologically active.

Human spermicidal activity of inorganic and organic oxidants.

OBJECTIVE: To identify prospective oxidants that rapidly immobilize human sperm upon contact with human semen. DESIGN: Inorganic, organic, and enzymatically-generated oxidants were mixed with human semen and spermicidal activity was tracked by a modified Sander-Cramer assay. SETTING: Commercial and university-based laboratories. PATIENT(S): Semen samples obtained through a university-based andrology laboratory. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Quantitation of spermicidal activity of test oxidants. RESULT(S): Sperm lost motility within 20 seconds of exposure to enzymatically generated free iodine (I(2)). Toluidine blue, phenazine methosulfate, or methylene blue exhibited some, albeit much less, spermicidal activity. Oxidants formed by mixing ascorbic acid with Fe(III)-EDTA, xanthine with xanthine oxidase, or by exposing sperm to the nitric oxide generator, SIN-1 (3-morpholinosydnonimine hydrochloride), were far less potent spermicidal agents. CONCLUSION(S): Free I(2) formed in situ and presented to semen is an extremely potent spermicide. Additional studies on methods of generating de novo I(2) may be beneficial in developing a novel new class of nondetergent-based spermicides.

Early CK-MB elevations predict ischemic events in stable chest pain patients.

OBJECTIVE: To demonstrate that creatine kinase-MB fraction (CK-MB) elevations within three hours of presentation in the emergency department (ED) are associated with subsequent ischemic events in clinically stable chest pain patients. METHODS: Prospective cohort study at two university- affiliated teaching hospitals. Participants were consenting ED chest pain patients 25 years old or older without evidence of rhythm or hemodynamic instability (n = 449). Exclusions included ST-segment elevation > or = 0.1 mV in > or = 2 electrocardiogram leads, chest wall trauma, abnormal x-ray studies, and incomplete data collection. Measurements included presenting and three-hour CK-MB levels, presenting ECG, initial clinical impression of coronary care unit need, and clinical follow up. Monitored adverse events included myocardial ischemia necessitating coronary angioplasty or cardiac bypass surgery, recurrent in-hospital myocardial infarction, bradycardia requiring pacing, emergent cardioversion, cardiogenic shock, ventricular fibrillation, and death. RESULTS: Overall, nine (2%) of 449 patients experienced an ischemic event within the first 48 hours. All nine patients required either coronary angioplasty or bypass surgery. Four (44%) of the nine patients with 48-hour ischemic events had elevated CK-MB levels. Of 23 patients who had complications within one week of ED presentation, seven (30%) had elevated ED CK-MB levels. An elevated CK-MB level was associated with an ischemic event both within 48 hours (risk ratio 9.5; 95% CI 2.7-33.7) and within one week (risk ration 5.2; 95% CI 2.3-11.7). CONCLUSIONS: An elevated CK-MB level within three hours of ED presentation is associated with a subsequent ischemic event in the clinically stable chest pain patient without ST-segment elevation. However, the ED CK-MB identifies only a minority or otherwise low-risk patients who develop ischemic events; other markers for diagnosing myocardial ischemia in the ED are needed.

Diagnostic value of creatine kinase and creatine kinase MB isoenzyme in chronic hemodialysis patients: a longitudinal study.

Serial measurements of total creatine kinase (CK) and the MB isoenzyme of CK (CK-MB) were performed on 18 stable hemodialysis patients over a 36 month interval in a longitudinal study designed to examine CK and CK-MB variability. Total CK was measured by the DuPont ACA pack procedure. CK-MB was quantitated by the ACA CK-MB ion exchange pack procedure, and by electrophoresis. The mean CK level was 242 +/- 179 IU/l (+/- SD). The mean CK-MB level was approximately 8% of the total CK. Seventy-two percent of the patients had at least one elevated CK level during the course of this study. Depending upon the method of analysis, 88 to 100% of the patients had at least one elevated CK-MB level. Marked and sporadic variations in both total CK and CK-MB levels were apparent, but were not related to the assay. Patients receiving long-term intramuscular androgens had higher CK, but not CK-MB, than did hemodialysis patients not receiving these agents. Discontinuation of the intramuscular androgens for four weeks led to a fall in CK in only one of four patients. Therefore there appears to be a biological effect of exogenous androgens which may play a role in elevating CK in hemodialysis patients.

"Outcome-counting'-Significance tests from incomplete predictions of order.

Experiments testing models usually predict that some treatment effects will be greater than others, but they do not as a rule feel able to predict the exact ordering of treatment effects. A useful significance test for such incomplete order predictions can be obtained by comparing the number of possible outcomes (orderings) with the number that satisfy the predictions. The difficulty is to count the outcomes satisfying the predictions, which can be a lengthy task if there are many treatments. A straightforward method of outcome-counting is derived from simple algebra and examples of its use are given.

Comparison of the response of primary murine peritoneal macrophages and the U937 human histiocytic cell line to challenge with in vitro generated clinically relevant UHMWPE particles.

The response of primary murine macrophages and the U937 human histiocytic cell line to challenge with clinically relevant UHMWPE wear debris of known particle size and dose was evaluated. Particles with mean sizes of 0.24, 0.45, 1.71, 7.62 and 88 microm were co-cultured with cells for 24 hours prior to assessment of cell viability and production of the osteolytic mediators IL-1beta, IL-6, TNFalpha and, in supernatants from murine phagocytes, PGE2 and GM-CSF. All particle fractions were evaluated at particle volume (microm3) to cell number ratios of 10 : 1 and 100 : 1 (and, additionally, 0.1 : 1 and 1 : 1 for U937 cells). These ratios had previously been identified as the most stimulatory and clinically relevant. Although the results for the cell line were highly variable, stimulation with phagocytosable particles (range 0.1 to 15 microm) resulted in enhanced levels of cytokine secretion by both murine macrophages and U937 histiocytes. The most biologically active particles were sub-micrometre in size. However, U937 cells responded to wear debris at much lower particle volume to cell number ratios (>0.1 microm3 per cell) than the murine cells (> 10 microm3 per cell). No GM-CSF was produced by particle or LPS stimulated murine macrophages. Similarly, U937 histiocytes failed to secrete any IL-1beta. Neither macrophage population responded to stimulation with the largest (88 microm) particles. These results confirm earlier findings and suggest that the size of UHMWPE wear particles is a critical factor in macrophage activation. Moreover, primary murine macrophages have been demonstrated to be a suitable model for studying cell-particle interactions in vitro.

A reassessment of the product specificity of the NADPH:O2 oxidoreductase of human neutrophils.

Native ferricytochrome c, but not acetylated ferricytochrome c, stimulates the flow of electron equivalents passing through the neutrophil NADPH:O2 oxidoreductase complex. At 28 mM it increases NADPH oxidase activity by 157 +/- 15% (n = 5) over that measured in its absence. Enhanced activity is predominantly seen in oxidoreductase-rich 27,000 X g membrane preparations obtained from phorbol myristate acetate activated cells. Superoxide formation is also enhanced. Although some of the stimulatory activity seen with addition of native ferricytochrome c to oxidoreductase-rich membrane suspensions might have been explained in terms of mitochondrial contamination, this was ruled out. Comparable membrane preparations from resting cells were devoid of NADPH oxidase activity. Azide, a well-known inhibitor of the electron transport chain, did not block the enhancing effect of native ferricytochrome c. These results indicate that native ferricytochrome c is not a suitable scavenger of superoxide in quantitating the product specificity of the oxidoreductase since it amplifies the apparent rate of superoxide formation with respect to measured rates of NADPH oxidation conducted in its absence. By using acetylated ferricytochrome c in place of native ferricytochrome c in quantitating the product specificity of the oxidoreductase we show that no more than 70% of the electron equivalents donated by NADPH to the oxidoreductase are involved in superoxide formation. The remaining 30% of the electron equivalents given up by NADPH to the oxidoreductase appear to be involved in direct formation of hydrogen peroxide.

Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils.

Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate. Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions. Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex. Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns. Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex. When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.