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1.
Pediatr Obes ; 13(2): 103-110, 2018 02.
Article in English | MEDLINE | ID: mdl-27977909

ABSTRACT

BACKGROUND: Public health experts raise concerns about adolescents' and black youth's greater exposure to TV advertising for unhealthy foods and beverages compared with children and white youth. OBJECTIVES: Examine how television-viewing patterns and rates of advertising during targeted programming contribute to this greater exposure. METHODS: Nielsen panel data provided viewing times and amount of food advertising viewed on U.S. television in 2008 and 2012. Researchers compared results by network type (black-, child- and youth-targeted), age group (preschoolers, children and adolescents) and race (black and white youth). RESULTS: Food advertising exposure increased with age for both black and white youth, but black youth viewed approximately 50% or more ads than did white youth of the same age. Higher rates of food advertising on youth-targeted networks explained greater adolescent exposure. However, greater television viewing and higher rates of advertising on youth- and black-targeted networks both contributed to black youth's greater exposure. From 2008 to 2012, increases in food-ads-per-hour increased exposure for all youth. CONCLUSIONS: Food advertisers and networks, especially those targeting adolescents and black youth, must do more to reduce advertising that negatively impacts young people's health. Furthermore, reducing commercial-television viewing by black youth may help reduce health disparities affecting their communities.


Subject(s)
Advertising/statistics & numerical data , Black or African American/statistics & numerical data , Food , Television/statistics & numerical data , White People/statistics & numerical data , Adolescent , Advertising/methods , Beverages , Child , Child, Preschool , Humans , Male , Time Factors , United States
2.
Pediatr Obes ; 13(4): 256-264, 2018 04.
Article in English | MEDLINE | ID: mdl-29076259

ABSTRACT

BACKGROUND: Food companies often use healthy lifestyle messages in child-directed advertising, raising public health concerns about health halo effects for nutrient-poor food/drinks. OBJECTIVE: Examine effects of health messages promoting nutrient-poor foods in child-directed advertising. METHODS: Randomized controlled experiment (N = 138). Children (7-11 years) viewed three child-friendly commercials in one of three conditions: (1) health halo (unfamiliar nutrient-poor food/drink ads with healthy messages); (2) nutrient-poor food/drink ads with other messages and (3) healthy food/drink ads. They rated the commercials and advertised products, provided attitudes about exercise and nutrition and consumed and rated healthy and unhealthy snack foods. RESULTS: Children in the health halo condition rated the advertised nutrient-poor products as significantly healthier compared with children in other conditions (p = .003), but the other commercials did not affect children's attitudes about other advertised products (p's > .50). Child age, gender or TV viewing habits did not significantly predict their ratings (p's > .18). There was no evidence that healthy lifestyle messages and/or healthy food commercials improved children's attitudes about nutrition, exercise or healthy snack consumption. CONCLUSION: Promoting healthy lifestyle messages in child-directed commercials for nutrient-poor food/drinks likely benefits brands by increasing products' perceived healthfulness, but these ads are unlikely to positively affect children's attitudes about health and nutrition.


Subject(s)
Advertising , Attitude to Health , Diet, Healthy/psychology , Effect Modifier, Epidemiologic , Health Education/methods , Nutritive Value , Beverages , Child , Exercise , Female , Food , Health Knowledge, Attitudes, Practice , Humans , Male , Nutritional Status , Snacks , Television
3.
Technol Cancer Res Treat ; 13(6): 561-9, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24000980

ABSTRACT

Irreversible electroporation (IRE) has been shown to be an effective method of killing cells locally. In contrast to radiofrequency ablation, the mechanism by which cells are thought to die via IRE is the creation of pores in cell membranes, without substantial increase in tissue temperature. To determine the degree to which cell death is non-thermal, we evaluated IRE in porcine hepatocytes in vivo. Using pulse widths of 10 µs, bursts of 3 kV square-wave pulses were applied through a custom probe to the liver of an anesthetized pig. Affected tissue was evaluated histologically via stainings of hematoxylin & eosin (H&E), nitroblue tetrazolium (NBT) to monitor cell respiration and TUNEL to gauge apoptosis. Temperature was measured during the application of electroporation, and heat transfer was modeled via finite element analysis. Cell death was calculated via Arrhenius kinetics. Four distinct zones were observed within the ring return electrode; heat-fixed tissue, coagulation, necrotic, and viable. The Arrhenius damage integral estimated complete cell death only in the first zone, where the temperature exceeded 70°C, and partial or no cell death in the other zones, where maximum temperature was approximately 45°C. Except for a limited area near the electrode tip, cell death in IRE is predominantly due to a non-thermal mechanism.


Subject(s)
Cell Death , Electroporation , Finite Element Analysis , Algorithms , Animals , Cell Survival , Electroporation/methods , Liver/pathology , Swine
4.
Pediatr Obes ; 9(5): 362-72, 2014 Oct.
Article in English | MEDLINE | ID: mdl-23818245

ABSTRACT

BACKGROUND: Food marketing contributes to childhood obesity. Food companies commonly place display advertising on children's web sites, but few studies have investigated this form of advertising. OBJECTIVES: Document the number of food and beverage display advertisements viewed on popular children's web sites, nutritional quality of advertised brands and proportion of advertising approved by food companies as healthier dietary choices for child-directed advertising. METHODS: Syndicated Internet exposure data identified popular children's web sites and food advertisements viewed on these web sites from July 2009 through June 2010. Advertisements were classified according to food category and companies' participation in food industry self-regulation. The percent of advertisements meeting government-proposed nutrition standards was calculated. RESULTS: 3.4 billion food advertisements appeared on popular children's web sites; 83% on just four web sites. Breakfast cereals and fast food were advertised most often (64% of ads). Most ads (74%) promoted brands approved by companies for child-directed advertising, but 84% advertised products that were high in fat, sugar and/or sodium. Ads for foods designated by companies as healthier dietary choices appropriate for child-directed advertising were least likely to meet independent nutrition standards. CONCLUSIONS: Most foods advertised on popular children's web sites do not meet independent nutrition standards. Further improvements to industry self-regulation are required.


Subject(s)
Advertising , Choice Behavior , Feeding Behavior , Food Industry/legislation & jurisprudence , Internet , Pediatric Obesity/prevention & control , Adolescent , Advertising/legislation & jurisprudence , Beverages , Child , Fast Foods , Female , Food , Humans , Male , Nutritive Value , Pediatric Obesity/epidemiology , Policy Making , Social Marketing , Video Games
5.
Water Sci Technol ; 63(12): 2853-9, 2011.
Article in English | MEDLINE | ID: mdl-22049710

ABSTRACT

Microcystis aeruginosa was cultured in biologically treated municipal effluent to simulate blue-green algal bloom conditions in a treatment lagoon. The effect of algae in the early, mid and late phases of growth on membrane fouling, chemical coagulation (alum or aluminium chlorohydrate (ACH)) and hydraulic cleaning on the microfiltration of this effluent was investigated. The effect of M. aeruginosa in the early phase was negligible and gave a similar flux profile and permeate volume to that of effluent alone. The increase in M. aeruginosa concentration for the mid and late phases caused a significant reduction in permeate volume compared with the early phase. Full flux recovery was achieved with an alum dose of 1 mg Al3+ L(-1) (early phase) and 10 mg Al3+ L(-1) (mid phase), demonstrating that membrane fouling was hydraulically reversible. For the late phase, the highest flux recovery was 89%, which was achieved with an alum dose of 5 mg Al3+ L(-1). Higher alum dosages resulted in a reduction in flux recovery. The use of 1.5 pm pre-filtration after alum treatment showed little improvement in water quality but led to a drastic reduction in flux recovery, which was attributed to diminishing the protective layer on the membrane surface, thus enabling internal fouling. The performance of ACH was comparable to alum at low dissolved organic carbon (DOC) and cell concentration, but was not as effective as alum at high DOC and cell concentration due to the formation of more compact ACH flocs, which resulted in a higher cake layer specific resistance, leading to the deterioration of performance.


Subject(s)
Filtration , Harmful Algal Bloom , Membranes, Artificial , Microcystis/growth & development , Sewage/microbiology , Water Purification/methods , Aluminum Compounds/chemistry , Biodegradation, Environmental , Environmental Monitoring , Filtration/instrumentation , Filtration/methods , Water Purification/standards
6.
Water Sci Technol ; 62(9): 1975-83, 2010.
Article in English | MEDLINE | ID: mdl-21045321

ABSTRACT

Membrane fouling in microfiltration (MF) and ultrafiltration (UF) of an activated sludge (AS) effluent was investigated. It was found that the major membrane foulants were polysaccharides, proteins, polysaccharide-like and protein-like materials and humic substances. MF fouling by the raw effluent was governed by pore adsorption of particles smaller than the pores during the first 30 minutes of filtration and then followed the cake filtration model. UF fouling could be described by the cake filtration model throughout the course of filtration. Coagulation with alum and (poly)aluminium chlorohydrate (ACH) altered the MF fouling mechanism to follow the cake filtration model from the beginning of filtration. The MF and UF flux improvement by coagulation was due to the removal of some of the foulants in the raw AS effluent by the coagulants. The MF flux improvement was greater for alum than for ACH whereas the two coagulants performed equally well in UF. Coagulation also reduced hydraulically irreversible fouling on the membranes and this effect was more prominent in MF than in UF. The unified membrane fouling index (UMFI) was used to quantitatively evaluate the effectiveness of coagulation on membrane flux enhancement.


Subject(s)
Filtration/instrumentation , Filtration/methods , Sewage/chemistry , Waste Disposal, Fluid/instrumentation , Membranes, Artificial
7.
Water Sci Technol ; 62(7): 1682-8, 2010.
Article in English | MEDLINE | ID: mdl-20935388

ABSTRACT

Cyanobacterial blooms in the lagoons of sewage treatment plants can severely impact the performance of membrane plants treating the effluent. This paper investigates the impact of Microcystis aeruginosa in a secondary effluent on the microfiltration filterability and cleaning of the membrane. Alum coagulation and dissolved air flotation (DAF) were investigated to remove the algae and so enhance the volume of effluent processed, and their influence on reversible and irreversible fouling. Degree of fouling due to the algal components was found to be in decreasing order of algal cells, algal organic matter and extracellular organic matter. Alum coagulation with 5 mg L⁻¹ as Al³(+) led to a substantial increase in permeate volume, an increase in dissolved organic carbon removal, and a foulant layer which protected the membrane from internal fouling but which was hydraulically removable resulting in full flux recovery. Pre-treatment by DAF or 1.5 µm filtration following alum coagulation enhanced the flux rate and permeate volume but exposed the membrane to internal irreversible fouling.


Subject(s)
Filtration , Microcystis , Sewage/microbiology , Waste Management/methods , Alum Compounds , Membranes, Artificial , Spectrometry, Fluorescence
8.
Water Sci Technol ; 62(8): 1914-21, 2010.
Article in English | MEDLINE | ID: mdl-20962408

ABSTRACT

There are many water quality characteristics which could influence the filterability of biologically treated effluent from Melbourne's Western Treatment Plant (WTP). Statistical correlation was used to identify the key water characteristics affecting the microfiltration (MF) and ultrafiltration (UF) filterability in terms of permeate volume of the treated effluent. The models developed showed that turbidity, dissolved organic carbon (DOC) and total suspended solids (TSS) were the key factors which influenced the MF and UF filterability. Turbidity was the dominant factor affecting the accuracy of the model for MF filterability while DOC was the major factor affecting the accuracy of the model for UF filterability. A prediction accuracy of 85% was obtained for MF and 86% for UF filterability of the WTP effluent. The characteristics of the organic components of the wastewater were demonstrated by EEM spectra to have seasonal variation which would have reduced the prediction accuracy. As turbidity, DOC and TSS can be determined on-line, the models would be useful for rapid prediction of the filterability of WTP effluent and this may assist the control of low-pressure membrane filtration processes.


Subject(s)
Filtration , Membranes, Artificial , Models, Theoretical , Sewage/microbiology , Water Pollutants, Chemical/standards , Water Purification , Australia , Filtration/methods , Filtration/standards , Hydrodynamics , Linear Models , Pressure , Water Pollutants, Chemical/analysis , Water Purification/methods , Water Purification/standards
9.
Water Res ; 43(16): 3940-7, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19656542

ABSTRACT

Greywater treatment by UVC/H(2)O(2) was investigated with regard to the removal of chemical oxygen demand (COD). A COD reduction from 225 to 30 mgl(-1) (overall removal of 87%) was achieved after settling overnight and subsequent irradiation for 3h with 10mM H(2)O(2). Most of the contaminants were removed by oxidation since only 13% COD was removed by settlement. The removal of COD in the greywater followed a second-order kinetic equation, r=0.0637[COD][H(2)O(2)], up to 10mM H(2)O(2). A slightly enhanced COD removal was observed at the initial pH of 10 compared with pH 3 and 7. This was attributed to the dissociation of H(2)O(2) to O(2)H(-). The treatment was not affected by total concentration of carbonate (c(T)) of at least 3 mM, above which operation between pH 3 and 5 was essential. The initial biodegradability of the settled greywater (as BOD(5):COD) was 0.22. After 2h UVC/H(2)O(2) treatment, a higher proportion of the residual contaminants was biodegradable (BOD(5):COD=0.41) which indicated its potential as a pre-treatment for a biological process.


Subject(s)
Hydrogen Peroxide/chemistry , Ultraviolet Rays , Waste Disposal, Fluid/methods , Anions/analysis , Bicarbonates/analysis , Carbonates/analysis , Chlorides/analysis , Hydrogen Peroxide/analysis , Hydrogen-Ion Concentration , Kinetics , Oxygen/analysis , Photochemical Processes , Water Pollutants/analysis
10.
J Biomech Eng ; 126(4): 492-7, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15543867

ABSTRACT

Most soft tissues that are treated clinically via heating experience multiaxial states of stress and strain in vivo and are subject to complex constraints during treatment. Remarkably, however, there are no prior data on changes in the multiaxial mechanical behavior of a collagenous tissue subjected to isometric constraints during heating. This paper presents the first biaxial stress-stretch data on a collagenous membrane (epicardium) before and after heating while subjected to various biaxial isometric constraints. It was found that isometric heating does not allow the increase in stiffness at low strains that occurs following isotonic heating. Moreover increasing the degree of stretch prior to heating increased the thermal stability of the tissue consistent with the concept that mechanical loading primarily affects the activation entropy, not the activation energy.


Subject(s)
Hot Temperature , Models, Cardiovascular , Pericardium/physiology , Pericardium/radiation effects , Weight-Bearing/physiology , Animals , Biomechanical Phenomena/methods , Cattle , Computer Simulation , Elasticity , Stress, Mechanical , Tissue Culture Techniques
11.
Cell Mol Biol (Noisy-le-grand) ; 49(7): 1167-75, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14682400

ABSTRACT

This study tested an intervention to reduce sexual risk behaviors in a high risk impaired population: homeless African-American, Caucasian and Hispanic men with mental illness. In a comparison group clinical trial, men were assigned to an experimental cognitive-behavioral or a control intervention and followed up over 16 months. Men were recruited from a psychiatric program in two shelters for homeless men in Nashville, Tennessee. An ethnically mixed cohort of subjects (54% African-American, 42% Caucasian and 4% Hispanic) were included in the study. Most had a chronic psychiatric disorder and a co-morbid substance abuse disorder. The 257 participants who were sexually active (130 experimental, 127 control) prior to the trial were the main target of the intervention. An experimental intervention (SexG), adapted from Susser and Associates (51), comprised 6 group sessions. The control intervention was a 6-session HIV educational program. Sexual risk behavior was the primary outcome. The experimental and control groups were compared with respect to the mean score on a sexual risk index. Complete follow-up data were obtained on 257 men (100%) for the initial six-month follow-up. These individuals have been followed for the remainder of the 16-month follow-up. This intervention, (SexG), successfully reduced sexual risk behaviors of homeless mentally ill African-American, Caucasian and Hispanic men. Similar approaches may be effective in other impaired high-risk populations.


Subject(s)
Acquired Immunodeficiency Syndrome/prevention & control , Behavior Therapy , Ill-Housed Persons/psychology , Mental Disorders/complications , Sexual Behavior/psychology , Acquired Immunodeficiency Syndrome/complications , Black or African American , Humans , Male
12.
J Biomech Eng ; 125(4): 540-8, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12968579

ABSTRACT

Thermal denaturation can induce marked changes in the optical and mechanical properties of collagenous tissues. The optical properties are important in both therapeutic and diagnostic applications of lasers in medicine. Although mechanical stress can be caused by collagen shrinkage in laser-based therapies, how the mechanical loading state affects the optical properties is not well understood. We used a new computer-controlled biaxial testing system to subject bovine epicardium to various loading conditions both before and after multiple levels of thermal damage. An integrating sphere technique was used to measure transmittance and diffuse reflectance, from which absorption and scattering coefficients were calculated using a Monte Carlo method. Results showed that the scattering coefficient increased with increasing mechanical load but decreased as the degree of thermal damage increased. There was no significant change in the absorption coefficient due to thermal damage over the ranges studied.


Subject(s)
Burns/physiopathology , Culture Techniques/methods , Hot Temperature/adverse effects , Pericardium/physiopathology , Pericardium/radiation effects , Photometry/instrumentation , Tomography, Optical Coherence/methods , Animals , Burns/etiology , Connective Tissue/injuries , Connective Tissue/physiopathology , Connective Tissue/radiation effects , Culture Techniques/instrumentation , Dose-Response Relationship, Radiation , Elasticity , Equipment Failure , Pericardium/injuries , Photometry/methods , Swine , Temperature
13.
J Biomech Eng ; 125(3): 381-8, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12929243

ABSTRACT

Recent isothermal biaxial isotonic tests suggest that increasing the temperature hastens the rate of denaturation of epicardium whereas increasing the mechanical load during heating delays this process, findings that are consistent with prior uniaxial tests on tendons. Yet, contrary to uniaxial reports, a clear time-temperature-load equivalency was not found in this multiaxial setting. There is, therefore, a need to delineate multiaxial thermomechanical behavior in greater detail, and ultimately, to correlate changes therein with the underlying microstructure. Toward this end, we describe a new experimental approach for quantifying heating-induced changes in the multiaxial mechanical response of thin sheet-like specimens. Illustrative results are presented for bovine epicardium subjected to nine different thermomechanical loading protocols. Among other results, it is shown that thermal damage tends to increase the stiffness at low strains and that overall changes in extensibility correlate well with the degree of thermal damage independent of the specific thermomechanical protocol. Multiaxial changes in behavior are nevertheless complex, and there is a need for significantly more testing before constitutive relations can be formulated.


Subject(s)
Burns/etiology , Burns/physiopathology , Hot Temperature/adverse effects , Mechanotransduction, Cellular/radiation effects , Models, Cardiovascular , Pericardium/physiopathology , Pericardium/radiation effects , Weight-Bearing , Adaptation, Physiological , Animals , Anisotropy , Cattle , Culture Techniques , Elasticity , Stress, Mechanical
14.
J Biol Chem ; 276(37): 34941-7, 2001 Sep 14.
Article in English | MEDLINE | ID: mdl-11438529

ABSTRACT

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.


Subject(s)
Isoenzymes/metabolism , Serine Endopeptidases/metabolism , Humans , Pichia/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/pharmacology , Substrate Specificity , Tryptases
15.
Chem Biol ; 8(12): 1131-41, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11755392

ABSTRACT

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.


Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Amino Acid Sequence , Enzyme Activation , Histocompatibility Antigens Class I/metabolism , Humans , Ligands , Molecular Sequence Data , Peptide Library , Proteasome Endopeptidase Complex , Structure-Activity Relationship , Substrate Specificity
16.
Water Res ; 35(18): 4455-63, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11763048

ABSTRACT

Natural organic matter (NOM) plays a significant role in fouling microfiltration membranes in drinking water treatment processes even though the NOM is retained only to a small extent. The aim of this study was to obtain a better understanding of the interactions between the fractional components of NOM and microfiltration membranes. Filtration experiments were performed using 0.22 microm hydrophobic and hydrophilic polyvinylidene fluoride (PVDF) membranes in a stirred-cell system on the NOM isolated from three Australian surface waters. As expected, the fouling rate for the hydrophobic membrane was considerably greater than for the hydrophilic membrane. Focusing on the hydrophobic membrane, it was shown that the high molecular weight fraction of NOM ( > 30 kDa) was responsible for the major flux decline. Filtration tests on the four fractions of NOM isolated on the basis of hydrophobicity and charge using non-functionalised and anionic resins revealed that the fouling potential for the three waters was hydrophilic neutral > hydrophobic acids > transphilic acids > hydrophilic charged. The low-aromatic hydrophilic neutral compounds were the main determinant of the rate and extent of flux decline. This was linked to the colloidal size fraction ( > 30 kDa) and to the selective concentration of calcium in the fraction leading to organics-Ca2+ bridging. It was also shown that the higher the aromaticity of the NOM the greater the flux decline, and the aromatics mainly resided in the hydrophobic acids fraction. Overall, the fouling mechanism controlling the flux decline involved the combined effects of adsorptive and colloidal fouling by the hydrophilic neutral fraction in the internal pore structure of the membrane.


Subject(s)
Membranes, Artificial , Organic Chemicals/analysis , Water Purification/methods , Adsorption , Colloids , Filtration , Quality Control , Resins, Plant/chemistry , Water Movements
17.
Nat Struct Biol ; 7(9): 762-5, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10966646

ABSTRACT

Granzyme B is a serine protease of the chymotrypsin fold that mediates cell death by cytotoxic lymphocytes. It is a processing enzyme, requiring extended peptide substrates containing an Asp residue. The determinants that allow for this substrate specificity are revealed in the three-dimensional structure of granzyme B in complex with a macromolecular inhibitor. The primary specificity for Asp occurs through a side-on interaction with Arg 226, a buried Arg side chain of granzyme B. An additional nine amino acids make contact with the substrate and define the granzyme B extended substrate specificity profile. The substrate determinants found in this structure are shared by other members of this protein class and help to reveal the properties that define substrate specificity.


Subject(s)
Apoptosis , Bacterial Proteins/metabolism , Escherichia coli Proteins , Periplasmic Proteins , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Aspartic Acid/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , Glycosylation , Granzymes , Humans , Models, Molecular , Mutation , Protein Conformation , Rats , Sequence Alignment , Serine Endopeptidases/genetics , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/genetics , Substrate Specificity
18.
J Biol Chem ; 275(34): 26333-42, 2000 Aug 25.
Article in English | MEDLINE | ID: mdl-10831593

ABSTRACT

Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-(X)P2-(Ser)P1-(Arg)P1'-(Ala)) and (P4-(X)P3-(Arg/Lys)P2-(Ser)P1(Arg) P1'(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 nm but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nm. Single-chain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nm), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events.


Subject(s)
Receptors, Thrombin/metabolism , Serine Endopeptidases/metabolism , Urokinase-Type Plasminogen Activator/metabolism , Animals , Binding Sites , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/metabolism , HeLa Cells , Humans , Models, Molecular , Rabbits , Receptor, PAR-2 , Structure-Activity Relationship , Substrate Specificity , Tumor Cells, Cultured , Xenopus
19.
Proc Natl Acad Sci U S A ; 97(14): 7754-9, 2000 Jul 05.
Article in English | MEDLINE | ID: mdl-10869434

ABSTRACT

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137, 180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.


Subject(s)
Combinatorial Chemistry Techniques , Coumarins/metabolism , Endopeptidases/metabolism , Fluorescent Dyes/metabolism , Peptides/chemical synthesis , Peptides/metabolism , Coumarins/chemistry , Cysteine Endopeptidases/metabolism , Serine Endopeptidases/metabolism , Substrate Specificity
20.
Genetics ; 154(2): 777-85, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10655229

ABSTRACT

Recently developed phylogeographic analyses that incorporate genealogical relationships of alleles offer the exciting prospect of disentangling historical from contemporary events. However, the relative advantages and shortfalls of this approach remain to be studied. We compared the nested cladistic method to the more traditional analysis of variance approach in a study of intraspecific genetic variation in the freshwater mussel, Lampsilis hydiana. We surveyed 257 specimens for nucleotide sequence level variation in a fragment of the mitochondrial 16S rRNA gene. When compared side by side, nested cladistic analysis and analysis of molecular variance (AMOVA) identified fragmentation of Arkansas river populations from remaining populations to the southwest. Nested cladistic analysis identified a second, more recent separation of Ouachita and Upper Saline river populations that was not detected by AMOVA. Differences among analytical methods probably arise from treatment of spatial hierarchical information: hierarchical groups emerge via a parsimony criterion in nested cladistic analysis but must be specified a priori in AMOVA. Both methods identified significant genetic structure among localities within hierarchical groups. Results from AMOVA suggested little gene flow among local populations with an island model. However, inferences about process that gave rise to patterns at this level were not possible in nested cladistic analysis, because an ancestral (interior) haplotype was not observed for a key one-step clade in the parsimony network. Our results suggest that, under some circumstances, nested cladistic analysis has lower power than more traditional analysis of variance to infer processes at the local population level.


Subject(s)
Bivalvia/genetics , Genetic Variation , Analysis of Variance , Animals , Base Sequence , Bivalvia/classification , DNA Primers , Haplotypes , Phylogeny , RNA, Ribosomal, 16S/genetics
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