bluuebunny/crossref_metadata_2025 huggingface dataset:
In situ remediation of MTBE utilizing ozone
Authors: Dey Jeffrey C. , Rosenwinkel Paul , Wheeler Kevin
Published: January 2003

There has been a great deal of focus on methyl tertiary butyl ether (MTBE) over the past few years by
local, state, and federal government, industry, public stakeholders, the environmental services market, and
educational institutions. This focus is, in large part, the result of the widespread detection of MTBE in
groundwater and surface waters across the United States. The presence of MTBE in groundwater has been attributed
primarily to the release from underground storage tank (UST) systems at gasoline service stations.
MTBE's physical and chemical properties are different than other constituents of gasoline that have
traditionally been cause for concern [benzene, toluene, ethylbenzene, and xylenes (BTEX)]. This
difference in properties is why MTBE migrates differently in the subsurface environment and exhibits different
constraints relative to mitigation and remediation of MTBE once it has been released to subsurface soils and
groundwater.

Resource Control Corporation (RCC) has accomplished the remediation of MTBE from subsurface soil and
groundwater at multiple sites using ozone. RCC has successfully applied ozone at several sites with different
lithologies, geochemistry, and concentrations of constituents of concern. This article presents results from
several projects utilizing in situ chemical oxidation with ozone. On these projects MTBE
concentrations in groundwater were reduced to remedial objectives usually sooner than anticipated. © 2002
Wiley Periodicals, Inc.
Directionally drilled horizontal wells offer cost savings and technical advantages over alternative soil and groundwater remediation systems
Authors: Fournier Louis B.
Published: January 2003

Directionally drilled horizontal wells offer the opportunity for significant cost savings and technical
advantages over alternative trenched well and vertical well soil and groundwater remediation systems in many cases.
The magnitude of the cost savings is a function of the remediation technology deployed and the values placed on the
reduction of site impacts, dramatic reduction in the time required to achieve site remediation goals and
requirements, the ability of horizontal well remediation to easily treat normally recalcitrant contaminants such as
MTBE, and the ability to drill under paved areas, operating plants, residential areas, landfills, lagoons,
waterways, ponds, basins, and other areas that are normally difficult or impossible to access with conventional
drilling or trenching methods. In addition to improvements in site access capabilities, horizontal wells have been
found capable of addressing contaminants that vertical wells do not readily treat, even with the same remediation
technology deployed, especially if air‐based remediation technologies are deployed. With biosparging, for
example, greater treatment capabilities of horizontal wells over vertical wells are attributed to greater oxygen
flux over a broader area, a larger treatment zone, and extremely prolonged residence of groundwater contaminants in
the aerobic treatment area, typically months or years. This article describes the use of directionally drilled
horizontal wells for application of a variety of treatment technologies and includes costs of various options with a
detailed comparison of biosparging options. © 2002 Wiley Periodicals, Inc.
Activity and Use Limitations applied to Risk‐Based Corrective Action—A checklist for efficacy
Authors: Sonnenfeld Paul
Published: January 2003

Activity and Use Limitations (AULs) comprise both institutional controls and engineering controls.
AULs, when properly implemented, are valuable tools in the risk‐based corrective action arsenal.
However, the proponent of AULs must evaluate their life‐cycle costs and the short‐term and
long‐term effectiveness of the AULs to protect human health and the environment. This article describes
the various types of AULS and provides a checklist for evaluating the efficacy of the proposed AULs. ©
2002 Wiley Periodicals, Inc.
Incorporating ecology and ecological risk into long‐term stewardship on contaminated sites
Authors: Burger Joanna
Published: January 2003

The long‐term management of environmental contamination will be a major activity at many sites in the
foreseeable future. While human health issues often drive decisions about cleanup, restoration, and
long‐term stewardship, ecological considerations are also major driving forces and are of paramount
importance to the public. Incorporation of ecological considerations into decisions about environmental
protection, both short term and long term, requires (1) understanding public perceptions of ecological
values, including aesthetics and existence values, (2) understanding contamination issues within a
context of the structure and functioning of ecosystems, (3) developing bioindicators of ecological
health (including human), (4) developing indicators of ecosystem functioning, and
(5) developing and implementing a biomonitoring plan before, during, and after remediation so that
adverse effects can be ascertained before they become irreversible. Both remediation/restoration and
long‐term stewardship goals must be informed by public policy mandates that include public participation
and healthy human and ecological systems. This article examines these issues as they relate to
cost‐effective, long‐term protection of human and ecological health on contaminated lands. ©
2002 Wiley Periodicals, Inc.
Phytoremediation of industrial sludge using vegetation‐assisted dewatering
Authors: Qiu Xiujin , Loehr Raymond C.
Published: January 2003

With the emergence of risk‐based corrective action decisions, there is interest in the use of more
natural techniques that may be as protective as the traditional removal, landfill, or capping approaches for
impoundment closure. The use of phytoremediation is one of the more promising techniques. This article presents
the results of a three‐year field‐pilot phytoremediation study that involved the use of plants to
enhance sludge dewatering at an inactive natural gas‐cracking wastewater lagoon. The dewatering was
accompanied by contaminant reduction of benzene, toluene, xylene (BTX), and naphthalene concentrations
to below the cleanup goals. Meanwhile, the concentration reductions of three or more ring polynuclear aromatic
hydrocarbons (PAHs) varied between 30 percent and 60 percent, except for
dibenz[a,h]anthracene. The residual PAHs in the sludge are not leaching. Parallel laboratory studies
suggest a reduced PAH availability and mobility in the unsaturated zone sludge. © 2002 Wiley Periodicals,
Inc.
Kinetics of biochemically driven metal precipitation in synthetic landfill leachate
Authors: Samaranayake Rajika , Singhal Neresh , Lewis Gillian , Hyland Margaret
Published: January 2003

This study investigates the effectiveness of using metal sulphide and carbonate precipitation mechanisms
combined with a landfill‐derived mixed bacterial population. The study was conducted under controlled
substrate conditions in anaerobic batch reactors. High chemical oxygen demand (COD):sulphate ratios,
butyrate, propionate, and acetate were used anaerobically by bacteria for growth with associated sulphate
reduction as well as sulphide and carbonate generation. Propionate and butyrate degradation occurred during
sulphate reduction by sulphate‐reducing bacteria while acetate degradation was associated with
methanogenesis by methanogenic bacteria. Using low COD, sulphate ratios showed limited acetate utilization, but
sulphate reduction still occurred. Precipitation of Cd, Cu, Zn, Ni, and Fe sulphides occurred quickly and was
completed in 15 to 30 days, while Ca, Mn, and Mg carbonates formed after 40 to 50 days and some soluble metal
remained even after 120 days. The rate of metal precipitation was in the order of
Cd>Cu>Zn>Ni>Fe>Mn>Mg>Ca. Bacterially mediated metal precipitation occurred slower than that
recognized for chemical precipitation. These findings suggest that contaminant transport models based on chemical
equilibrium metal behaviors may over‐predict metal removal by bio‐precipitation. © 2002 Wiley
Periodicals, Inc.
Non‐inferiority trials: design concepts and issues – the encounters of academic consultants in statistics
Authors: D'Agostino Ralph B. , Massaro Joseph M. , Sullivan Lisa M.
Published: January 2003

Placebo‐controlled trials are the ideal for evaluating medical treatment efficacy. They allow for control of the placebo effect and are most efficient, requiring the smallest numbers of patients to detect a treatment effect. A placebo control is ethically justified if no standard treatment exists, if the standard treatment has not been proven efficacious, there are no risks associated with delaying treatment or escape clauses are included in the protocol. Where possible and justified, they should be the first choice for medical treatment evaluation. Given the large number of proven effective treatments, placebo‐controlled trials are often unethical. In these situations active‐controlled trials are generally appropriate. The non‐inferiority trial is appropriate for evaluation of the efficacy of an experimental treatment versus an active control when it is hypothesized that the experimental treatment may not be superior to a proven effective treatment, but is clinically and statistically not inferior in effectiveness. These trials are not easy to design. An active control must be selected. Good historical placebo‐controlled trials documenting the efficacy of the active control must exist. From these historical trials statistical analysis must be performed and clinical judgement applied in order to determine the non‐inferiority margin M and to assess assay sensitivity. The latter refers to establishing that the active drug would be superior to the placebo in the setting of the present non‐inferiority trial (that is, the constancy assumption). Further, a putative placebo analysis of the new treatment versus the placebo using data from the non‐inferiority trial and the historical active versus placebo‐controlled trials is needed. Useable placebo‐controlled historical trials for the active control are often not available, and determination of assay sensitivity and an appropriate M is difficult and debatable. Serious consideration to expansions of and alternatives to non‐inferiority trials are needed. Copyright © 2003 John Wiley & Sons, Ltd.
Synthesis and Application of Starch 3, 5‐Dinitrobenzoate
Authors: Yu Jiugao , Yang Dongzhi
Published: January 2003

A novel starch ester was prepared by the reaction of starch and 3, 5‐dinitrobenzoyl chloride (DNBZ‐Cl). The product (starch 3, 5‐dinitrobenzoate, DNBZ‐ST) was characterized by means of elemental analysis, Fourier Transform Infrared (FTIR) and 13C NMR spectroscopy. The influences of molar ratio of DNBZ‐Cl to anhydroglucose (AGU), reaction temperature and time, and amount of pyridine on the degree of substitution (DS) were studied. The esterification reaction is essentially complete after 2 h. Increase in the molar ratio of DNBZ‐Cl to AGU leads to an increase in DS when the former varies from 2:1 to 5:1. DS increases with the reaction temperature when the latter is varied from 70 to 100 °C. DS first increases and then decreases with amount of pyridine, the highest DS was obtained when V(pyridine)/m(starch) was 53 mL/g. The thermal stability of DNBZ‐ST increases when the DS increases, and the degradation starts at 353 °C for the sample of DS 2.14. Creatinine is a toxin accumulated in the blood of chronic renal failure (CRF) patients and a special adsorbent for creatinine is not reported in literature. DNBZ‐ST displayed specific adsorption ability for creatinine, the adsorption equilibrium is reached after 4 h. The adsorption capacity increases with the DS of adsorbent and creatinine concentration. When the creatinine concentration is higher than 300 mg/L, concentration has no apparent effect on the adsorption capacity. As the temperature of the solution is varied from 19 to 49 °C, adsorption capacity first decreases and then increases, being lowest at 37 °C. The adsorption capacity first increases and then decreases as the pH value increases, and is highest at pH 8.The highest adsorption capacity obtained was 25 mg of creatinine per gram of adsorbent at 37 °C, pH 7 and a creatinine concentration of 100 mg/L. The study on the FTIR and UV‐VIS spectra suggested that some chemical reaction took place between the DNBZ‐ST and creatinine in buffer solution.
Thermal Behavior of Zein‐based Biodegradable Films
Authors: Wang Ying , Rakotonirainy Andrianaivo M. , Padua Graciela W.
Published: January 2003

In previous work, zein was plasticized with oleic acid to obtain flexible films. However, it was observed that film properties were affected by the preparation method employed. Our objective was to investigate the effect of processing methods on thermal behavior of zein films by differential scanning calorimetry (DSC). Films containing 41% oleic acid were prepared by casting ethanol solutions of its components on flat surfaces or by extrusion of zein resins prepared by cold water precipitation of ethanol solutions of zein and oleic acid. Extrusion was carried out in single‐screw or twin‐screw extruders. Zein films were finished by hot rolling or heated in a Carver press. DSC thermograms showed large oleic acid melting peaks for cast films, smaller peaks for resin films, and no apparent peaks for heat‐treated samples. It was suggested that the resin formation process enhanced zein‐oleic acid interactions and promoted plasticization. All samples showed glass transitions at low temperatures.
Canavalia ensiformis Tailing Starch: A Functional Source of Dietary Fiber
Authors: Novelo‐Cen Loraine , Betancur‐Ancona David
Published: January 2003

Physicochemical and functional characteristics of the tailing starch isolated from the legume Canavalia ensiformis were determined. Proximal composition was 2.86% protein, 4.05% fiber, 0.37% fat, 1.51% ash and 91.33% carbohydrates as nitrogen‐free extract. Total dietary fiber content was 21.3%, soluble fiber was 2.2%, and insoluble fiber was 19.1% as determined by the Prosky method. Amylose content as quantified by differential scanning calorimetry (DSC) was 39.35%. Gelatinization temperature range as determined by DSC was from 77.9 °C to 88.1 °C, with 81.6 °C being the peak temperature. Transition enthalpy was 10.1 J/g. Swelling power ranged from 4.36 g water/g starch at 60 °C to 11.87 g water/g starch at 90 °C. Water solubility, analyzed within the same temperature interval, ranged from 3.29% to 16.93%. Water absorption capacity was 4.22 g water/g starch at 60 °C and 10.33 g water/g starch at 90 °C. Syneresis in a 6% gel stored at 4 °C for 24 h was 25.66 mL water/50 mL gel. Clarity, expressed as transmittance, was 10.46%. Initial pasting temperature was 93 °C, breakdown was —22 Brabender Units (BU), consistency was ‐5 BU, and setback was 17 BU.
Cyanoethylation of Cassia tora Gum
Authors: Sharma Brij Raj , Kumar Vineet , Soni Purshottam L.
Published: January 2003

Cyanoethylation of Cassia tora gum was carried out with acrylonitrile in presence of sodium hydroxide under different reaction conditions. Variables studied were concentrations of sodium hydroxide and acrylonitrile, reaction temperature and time. The degree of substitution, reaction efficiency and total extent of etherification were determined. The optimum conditions for preparing cyanoethyl Cassia tora gum (DS 0.44) were 0.608 mol acrylonitrile and 0.625 mol sodium hydroxide at 30 °C for 4 h. Rheological properties of cyanoethyl Cassia tora gum solutions showed non‐Newtonian pseudoplastic behavior regardless of the degree of substitution (DS). At a constant rate of shear the apparent viscosity of cyanoethyl Cassia tora gum solutions increased with the increase in %N of the product.
Non‐contact Atomic Force Microscopy of Starch Granules Surface. Part II. Selected Cereal Starches
Authors: Juszczak Lesław , Fortuna Teresa , Krok Franciszek
Published: January 2003

Atomic Force Microscopy (non‐contact method) was used to examine the surface of starch granules of selected cereals. Depressions which might be typical surface pores or ends of penetrating channels were observed in case of all examined granules. The size and shape of these depressions depended on the botanical origin of the starch. Regions of relatively flat surface without visible structures, pores and protrusions could be detected on the granules studied.
Non‐contact Atomic Force Microscopy of Starch Granules Surface. Part I. Potato and Tapioca Starches
Authors: Juszczak Lesław , Fortuna Teresa , Krok Franciszek
Published: January 2003

Atomic Force Microscopy (non‐contact method) was used in the present study to investigate the surface of potato and tapioca starch granules. Surface structures and pores or protrusions were observed both in the case of potato and tapioca starches. It was observed that tapioca starch granules seem to be smoother than those of potato starch, however also in the former case surface structures and protrusions were found. Regions of relatively flat surface without visible structures, pores and protrusions could be detected on the granules studied.
Further characterization of the putative human isopeptidase T catalytic site
Authors: Lacombe Thierry , Gabriel Jean-Marc
Published: January 2003

The human isopeptidase T (isoT) is a zinc‐binding deubiquitinating enzyme involved in the disassembly of free K48‐linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site‐directed mutagenized. None of the mutants were able to cleave a peptide‐linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48‐linked isopeptide ubiquitin dimer, which is an isoT‐specific substrate that mimics the K48‐linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48‐linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.
Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf‐1 or B‐Raf
Authors: Nadal-Wollbold Florence , Pawlowski Marc , Lévy-Toledano Sylviane , Berrou Eliane , Rosa Jean-Philippe , Bryckaert Marijke
Published: January 2003

Extracellular signal‐regulated kinase (ERK) activation pathways have been well characterized in a number of cell types but very few data are available for platelets. The thrombin‐induced signaling pathway leading to ERK2 activation in platelets is largely uncharacterized. In this study, we investigated the kinases involved in thrombin‐induced ERK2 activation in conditions of maximal ERK2 activation. We found that thrombin‐induced mitogen‐activated protein kinase/ERK kinase (MEK)1/2 activation was necessary for ERK2 phosphorylation. We obtained strong evidence that conventional protein kinase Cs (PKCs) and calcium are involved in thrombin‐induced ERK2 activation. First, ERK2 and MEK1/2 phosphorylation was totally inhibited by low concentrations (1 μM) of RO318425, a specific inhibitor of conventional PKCs. Second, Ca2+, from either intracellular pools or the extracellular medium, was necessary for ERK2 activation and conventional PKC activation, excluding the involvement of a new class of calcium‐insensitive PKCs. Third, LY294002 and wortmannin had no significant effect on ERK2 activation, even at concentrations that inhibit phosphatidylinositol (PI)3‐kinase (5 μM to 25 μM and 50 nM, respectively). This suggests that PI3‐kinase was not necessary for ERK2 activation and therefore, that PI3‐kinase‐dependent atypical PKCs were not involved. Surprisingly, in contrast to proliferative cells, we found that the serine/threonine kinases Raf‐1 and B‐Raf were not an intermediate kinase between conventional PKCs and MEK1/2. After immunoprecipitation of Raf‐1 and B‐Raf, the basal glutathione S‐transferase–MEK1 phosphorylation observed in resting platelets was not upregulated by thrombin and was still observed in the absence of anti‐Raf‐1 or anti‐B‐Raf antibodies. In these conditions, the in vitro cascade kinase assay did not detect any MEK activity. Thus in platelets, thrombin‐induced ERK2 activation is activated by conventional PKCs independently of Raf‐1 and B‐Raf activation.
Identification of 30 protein species involved in replicative senescence and stress‐induced premature senescence
Authors: Dierick Jean-François , Kalume Dário E , Wenders Frédéric , Salmon Michel , Dieu Marc , Raes Martine , Roepstorff Peter , Toussaint Olivier
Published: January 2003

Exposure of human proliferative cells to subcytotoxic stress triggers stress‐induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress‐induced premature senescence indicates that, at the level of protein expression, stress‐induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress‐induced premature senescence. These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways.
Endogenous plasma membrane t‐SNARE syntaxin 4 is present in rab11 positive endosomal membranes and associates with cortical actin cytoskeleton
Authors: Band Arja M , Ali Heidi , Vartiainen Maria K , Welti Saara , Lappalainen Pekka , Olkkonen Vesa M , Kuismanen Esa
Published: January 2003

Membrane fusion requires the formation of a complex between a vesicle protein (v‐SNARE) and the target membrane proteins (t‐SNAREs). Syntaxin 4 is a t‐SNARE that, according to previous overexpression studies, is predominantly localized at the plasma membrane. In the present study endogenous syntaxin 4 was found in intracellular vesicular structures in addition to regions of the plasma membrane. In these vesicular structures syntaxin 4 colocalized with rab11, a marker of recycling endosomes. Furthermore, syntaxin 4 colocalized with actin at the dynamic regions of the plasma membrane. Treatment with N‐ethylmaleimide, the membrane transport inhibitor, caused an increased accumulation of syntaxin 4/rab11 positive vesicles in actin filament‐like structures. Finally, purified recombinant syntaxin 4 but not syntaxin 2 or 3 cosedimented with actin filaments in vitro, suggesting direct interaction between these two proteins. Taken together, these data suggest that syntaxin 4 regulates secretion at the actin‐rich areas of the plasma membrane and may be recycled through rab11 positive intracellular membranes.
An enhancer complex confers both high‐level and cell‐specific expression of the human type X collagen gene
Authors: Chambers David , Young David A. , Howard Charles , Thomas J.Terrig , Boam David S. , Grant Michael E. , Wallis Gillian A. , Boot-Handford Raymond P.
Published: January 2003

Type X collagen expression is restricted to hypertrophic chondrocytes in the endochondral growth plate. Transient transfection of reporter constructs containing the human collagen X promoter into primary growth plate chondrocytes identified a cis‐acting positive regulatory DNA element(s) that has cell‐specific enhancer properties and binds a nuclear protein expressed specifically in growth plate chondrocytes. Functional disruption of this region results in a significant reduction in the activation of reporter gene transcription. The identified enhancer is a major element controlling both high‐level and cell‐specific expression of type X collagen gene.
Identification of the molecular chaperone alpha B‐crystallin in demineralized bone powder and osteoblast‐like cells
Authors: Behnam Keyvan , Murray Samuel S. , Whitelegge Julian P. , Brochmann Elsa J.
Published: January 2003

Bone is subjected to a variety of physiological, as well as cell‐deforming biomechanical stresses, including hydrostatic compression and fluid flow. However, little is known about the molecular mechanisms that protect bone cells from mechanical, ischemic, or oxidative damage. Crystallins are 20 kD heat shock proteins that function as molecular chaperones. We tested the hypothesis that alpha B‐crystallin (αB‐crystallin), the most widely expressed vertebrate crystallin, is present in bone and osteoblast‐like cells. Noncollagenous proteins (NCPs) were extracted from human demineralized bone matrix with 4 M guanidine HCl containing 0.5 M CaCl2 and protease inhibitors, defatted, dialyzed against 0.2% (v/v) Triton X‐100 in 100 mM Tris‐HCI (pH 7.2) and water, centrifuged, and lyophilized. The NCPs were separated by 2D IEF/SDS‐PAGE. The two most abundant 20 kD spots, with apparent pIs of 7.85 and 7.42 in urea gels, were excised, subjected to matrix‐assisted laser desorption ionization/time‐of‐flight mass spectrometry, and identified as αB‐crystallins. Indirect immunofluorescence localized αB‐crystallin to the interphase nucleus, cytoskeleton and cytoplasm of proliferating MC3T3‐El mouse osteoblast‐like cells, as well as the cytoskeleton and cytoplasm of confluent cells. In conclusion, αB‐crystallin is present in bone and osteoblast‐like cells. We hypothesize that αB‐crystallin may play a role in protecting the osteoblast cytoskeleton from mechanical stress and may be important in modulating nuclear or cellular functions, such as transcription or apoptosis, as observed in other tissues.

Published by Elsevier Science Ltd. On behalf of Orthopaedic Research Society.
A comparison of within‐ and between‐day reliability of discrete 3D lower extremity variables in runners
Authors: Ferber R. , McClay Davis I. , Williams D. S. , Laughton C.
Published: January 2003

It is important to understand the day‐to‐day variability that is attributed to repositioning of markers especially when assessing a treatment effect or response over time. While previous studies have reported reliability of waveform patterns, none have assessed the repeatability of discrete points such as peak angles, velocities and angular excursions which are often used when making statistical and clinical comparisons. The purpose of this study was to compare the within‐ and between‐day variability of discrete kinematic, kinetic, and ground reaction force (GRF) data collected during running. Comparisons for 20 recreational runners were evaluated for within‐ and between‐day reliability of discrete 3D kinematic, kinetic, and GRF variables. The results indicated that within‐day comparisons were more reliable than between‐day. Joint angular velocity and angular excursion values were more reliable between‐days as compared to absolute peak angle measures and may be more useful in interpreting changes in treatment over time. Between‐day kinematic and kinetic sagittal plane values were more reliable than secondary plane values. Reliability of GRF data was greater than kinematic and kinetic data for between‐day comparisons.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
COX‐2 selective NSAID decreases bone ingrowth in vivo
Authors: Goodman Stuart , Ma Ting , Trindade Michael , Ikenoue Takashi , Matsuura Ippe , Wong Neal , Fox Nora , Genovese Mark , Regula Don , Smith R. Lane
Published: January 2003

Whether non‐steroidal anti‐inflammatory drug (NSAID)—induced suppression of bone ingrowth is due to cyclooxygenase‐1 (COX‐1) inhibition, cyclooxygenase‐2 (COX‐2) inhibition, or through a yet unidentified pathway is unknown. In this study, the effects of a non‐specific COX‐1 and COX‐2 inhibitor, versus a specific COX‐2 inhibitor on bone ingrowth and tissue differentiation are examined in vivo. Harvest chambers were implanted unilaterally in the tibiae of eight mature, New Zealand white rabbits. After a 6‐week period for osseointegration of the chamber, the following oral treatments were given for 4 weeks each, followed by a harvest in each case: drinking water with no NSAID (control 1), Naproxen sodium–a COX‐1 and COX‐2 non‐specific inhibitor at a dose of 110 mg/kg/day in the drinking water, drinking water with no NSAID (control 2), and Rofecoxib—a COX‐2 inhibitor at a dose of 12.5 mg/day inserted directly into the rabbit's mouth. Harvested specimens were snap frozen, cut into serial 6 μm sections and stained with hematoxylin and eosin for general morphological characterization, and alkaline phosphatase (osteoblast marker). Sections were also processed for immunoperoxidase staining using monoclonal antibodies to identify cells expressing the vitronectin receptor (osteoclast‐like cells). With drinking water alone, the percentage of bone ingrowth averaged 24.8 ± 2.9% and 29.9 ± 4.5% respectively. Naproxen sodium in the drinking water and oral Rofecoxib decreased bone ingrowth significantly (15.9 ± 3.3%, p = 0.031 and 18.5 ± 2.4%, p = 0.035 compared to drinking water respectively). Both Naproxen sodium (p = 0.026) and Rofecoxib (p = 0.02) decreased the number of CD51 positive osteoclast‐like cells per section compared with drinking water alone. Rofecoxib decreased the area of osteoblasts per section area (p = 0.014) compared to controls, although the value for Naproxen sodium did not reach statistical significance. The results of the present study suggest that bone formation is suppressed by oral administration of an NSAID which contains a COX‐2 inhibitor. COX‐2 inhibitors currently taken for arthritis and other conditions may potentially delay fracture healing and bone ingrowth.

© 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Demonstration of functional M3‐muscarinic receptors in ventricular cardiomyocytes of adult rats
Authors: Pönicke Klaus , Heinroth‐Hoffmann Ingrid , Brodde Otto‐Erich
Published: January 2003

Muscarinic receptors (M‐receptors) in the mammalian heart are predominantly of the M2‐subtype. The aim of this study was to find out whether there might exist an additional myocardial non‐M2‐receptor.

For this purpose, we assessed, in adult rat isolated ventricular cardiomyocytes, carbachol‐induced [3H]‐inositol phosphate (IP) formation, and its inhibition by M‐receptor antagonists.

Carbachol (10−7–10−3 mol l−1) increased IP‐formation (maximal increase: 14±3% above basal, n=6). This increase was significantly enhanced by pretreatment with pertussis toxin (PTX, 250 ng ml−1 for 20 h): maximal increase was 31±5%, pEC50‐value was 5.08±0.33 (n=6).

In PTX‐pretreated cardiomyocytes 100 μmol l−1 carbachol‐induced IP‐formation was inhibited by atropine (pKi‐value: 8.89±0.10) and by the M3‐receptor antagonist darifenacin (pKi‐value: 8.67±0.23) but was not significantly affected by the M1‐receptor antagonist pirenzepine (1 μmol l−1) or the M2‐receptor antagonists AF‐DX 116 and himbacine (1 μmol l−1).

In conclusion, in adult rat cardiomyocytes there exists an additional, non‐M2‐receptor, that is coupled to activation of the phospholipase C/IP3‐pathway; this receptor is very likely of the M3‐subtype.

British Journal of Pharmacology (2003) 138, 156–160. doi:10.1038/sj.bjp.0704997
Regulation of muscarinic receptor function in developing oligodendrocytes by agonist exposure
Authors: Molina‐Holgado Eduardo , Khorchid Amani , Liu Hsueh‐Ning , Almazan Guillermina
Published: January 2003

Oligodendrocytes, the myelin forming cells in the CNS, express muscarinic acetylcholine receptors (mAChR), primarily M3, coupled to various signal transduction pathways.

In the present study we have investigated whether mAChR undergo functional agonist‐induced regulation in cultured oligodendrocyte progenitors and differentiated oligodendrocytes.

The muscarinic agonist, carbachol (CCh) caused a time‐dependent desensitization of phosphoinositide (PI) hydrolysis, and the internalization and down‐regulation of receptors. Short‐time desensitization (5 min) of PI hydrolysis occurred without receptor internalization and reached 54% by 1 h. The same treatment decreased cell surface receptors labelled with the non‐permeable ligand [3H]‐NMS by 47%, while total receptor density ([3H]‐scopolamine binding) decreased by 30%. Longer CCh treatment down‐regulated receptors by 70% and desensitized the PI response by 80%.

Although protein kinase C (PKC) activation desensitized mAChR, CCh‐mediated desensitization was independent of PKC.

Inhibition of receptor endocytosis by low temperature during the pre‐stimulation period or in the presence of hyperosmotic sucrose (0.5 M) blocked desensitization, receptor internalization and down‐regulation.

Recovery of surface mAChR and their functional activity following down‐regulation was slow, returning to control levels by 24 h after agonist removal. In progenitor cells, dose‐response curves for CCh‐mediated PI hydrolysis and c‐fos mRNA expression showed that newly synthesized mAChR were supersensitive after recovery.

Overall, the present results provide evidence of functional agonist‐mediated mAChR regulation in brain oligodendroglial cells.

British Journal of Pharmacology (2003) 138, 47–56. doi:10.1038/sj.bjp.0705002
Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5‐trisphosphate‐sensitive stores
Authors: Marshall Ian C B , Owen Davina E , Cripps Tim V , Davis John B , McNulty Shaun , Smart Darren
Published: January 2003

Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand‐gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores.

Human VR1‐transfected HEK293 cells (hVR1‐HEK293) were loaded with Fluo‐3 or a mixture of Fluo‐4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively.

In Ca2+‐free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1‐HEK293 cells (pEC50 6.45±0.05) but not in wild type cells. Capsaicin (100 μM) did not cause Ca2+ mobilization under these conditions.

RTX‐mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC50 5.84±0.04), the Ca2+ pump inhibitor thapsigargin (pIC50 7.77±0.04), the phospholipase C inhibitor U‐73122 (pIC50 5.35±0.05) and by depletion of inositol 1,4,5‐trisphosphate‐sensitive Ca2+ stores by pretreatment with the acetylcholine‐receptor agonist carbachol (20 μM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5‐trisphosphate‐sensitive Ca2+ stores in hVR1‐HEK293 cells.

In the presence of extracellular Ca2+, both capsaicin‐mediated and RTX‐mediated Ca2+ rises were a ttenuated by U‐73122 (10 μM, 30 min) and thapsigargin (1 μM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.

British Journal of Pharmacology (2003) 138, 172–176. doi:10.1038/sj.bjp.0705003
In vitro evidence for the presence of [3H]‐haloperidol uptake in rat brain
Authors: Ruiu Stefania , Marchese Giorgio , Saba Pier Luigi , Satta Rosalba , Gessa GianLuigi , Vaccari Andrea , Pani Luca
Published: January 2003

The neuroleptic [3H]‐haloperidol (HP) was taken up in synaptosomes prepared from rat brain, in a temperature‐, sodium ion‐, and energy‐dependent process.

The highest concentration of uptake sites (Vmax=2.37 pmol mg−1 protein min−1) was in the striatum with the other brain areas displaying lower (by 50–70%) values.

The affinity values (Km∼40 nM) were similar in all brain areas considered.

The pharmacological characterization did not indicate a well‐defined group of inhibitors, which suggested that HP might not use a transporter for recognized neurotransmitters.

The HP metabolites tested, including HPTP, were competitive inhibitors of [3H]‐HP uptake, an indirect indication that they may actively enter the striatal nerve endings through the same carrier.

Since the uptake process was partially affected by the incubation of [3H]‐HP in the presence of several antagonists of HP‐transforming cytochrome P450 isoforms, the binding of HP at some enzyme sites inside the synaptosome cannot be excluded.

In conclusion, the present results suggest that HP may be actively transported in the rat brain.

British Journal of Pharmacology (2003) 138, 188–192. doi:10.1038/sj.bjp.0705009
Characterization of the 5‐hydroxytryptamine receptors mediating contraction in the pig isolated intravesical ureter
Authors: Hernández Medardo , Barahona María Victoria , Simonsen Ulf , Recio Paz , Rivera Luis , Martínez Ana Cristina , García‐Sacristán Albino , Orensanz Luis M , Prieto Dolores
Published: January 2003

This study was designed to investigate the effect of 5‐hydroxytryptamine (5‐HT) and to characterize the 5‐HT receptors involved in 5‐HT responses in the pig intravesical ureter.

5‐HT (0.01–10 μM) concentration‐dependently increased the tone of intravesical ureteral strips, whereas the increases in phasic contractions were concentration‐independent. The 5‐HT2 receptor agonist α‐methyl 5‐HT, mimicked the effect on tone whereas weak or no response was obtained with 5‐CT, 8‐OH‐DPAT, m‐chlorophenylbiguanide and RS 67333, 5‐HT1, 5‐HT1A, 5‐HT3 and 5‐HT4 receptor agonists, respectively. 5‐HT did not induce relaxation of U46619‐contracted ureteral preparations. Pargyline (100 μM), a monoaminooxidase A/B activity inhibitor, produced leftward displacements of the concentration‐response curves for 5‐HT.

5‐HT‐induced tone was reduced by the 5‐HT2 and 5‐HT2A receptor antagonists ritanserine (0.1 μM) and spiperone (0.2 μM), respectively. However, 5‐HT contraction was not antagonized by cyanopindolol (2 μM), SDZ–SER 082 (1 μM), Y‐25130 (1 μM) and GR 113808 (0.1 μM), which are respectively, 5‐HT1A/1B, 5‐HT2B/2C, 5‐HT3, and 5‐HT4 selective receptor antagonists.

Removal of the urothelium did not modify 5‐HT‐induced contractions. Blockade of neuronal voltage‐activated sodium channels, α‐adrenergic receptors and adrenergic neurotransmission with tetrodotoxin (1 μM), phentolamine (0.3 μM) and guanethidine (10 μM), respectively, reduced the contractions to 5‐HT. However, physostigmine (1 μM), atropine (0.1 μM) and suramin (30 μM), inhibitors of cholinesterase activity, muscarinic‐ and purinergic P2‐receptors, respectively, failed to modify the contractions to 5‐HT.

These results suggest that 5‐HT increases the tone of the pig intravesical ureter through 5‐HT2A receptors located at the smooth muscle. Part of the 5‐HT contraction is indirectly mediated via noradrenaline release from sympathetic nerves.

British Journal of Pharmacology (2003) 138, 137–144. doi:10.1038/sj.bjp.0705019
Involvement of tachykinin receptors in Clostridium perfringens beta‐toxin‐induced plasma extravasation
Authors: Nagahama Masahiro , Morimitsu Shinsuke , Kihara Atsushi , Akita Masahiko , Setsu Koujun , Sakurai Jun
Published: January 2003

Clostridium perfringens beta‐toxin causes dermonecrosis and oedema in the dorsal skin of animals. In the present study, we investigated the mechanisms of oedema induced by the toxin.

The toxin induced plasma extravasation in the dorsal skin of Balb/c mice.

The extravasation was significantly inhibited by diphenhydramine, a histamine 1 receptor antagonist. However, the toxin did not cause the release of histamine from mouse mastocytoma cells.

Tachykinin NK1 receptor antagonists, [D‐Pro2, D‐Trp7,9]‐SP, [D‐Pro4, D‐Trp7,9]‐SP and spantide, inhibited the toxin‐induced leakage in a dose‐dependent manner. Furthermore, the non‐peptide tachykinin NK1 receptor antagonist, SR140333, markedly inhibited the toxin‐induced leakage.

The leakage induced by the toxin was markedly reduced in capsaicin‐pretreated mouse skin but the leakage was not affected by systemic pretreatment with a calcitonin gene‐related peptide receptor antagonist (CGRP8‐37).

The toxin‐induced leakage was significantly inhibited by the N‐type Ca2+ channel blocker, ω‐conotoxin MVIIA, and the bradykinin B2 receptor antagonist, HOE140 (D‐Arg‐[Hyp3, Thi5, D‐Tic7, Oic8]‐bradykinin), but was not affected by the selective L‐type Ca2+ channel blocker, verapamil, the P‐type Ca2+ channel blocker, ω‐agatoxin IVA, tetrodotoxin (TTX), the TTX‐resistant Na+ channel blocker, carbamazepine, or the sensory nerve conduction blocker, lignocaine.

These results suggest that plasma extravasation induced by beta‐toxin in mouse skin is mediated via a mechanism involving tachykinin NK1 receptors.

British Journal of Pharmacology (2003) 138, 23–30. doi:10.1038/sj.bjp.0705022
Strategies for helicase recruitment and loading in bacteria
Authors: Konieczny Igor
Published: January 2003

DNA replication initiation in prokaryotes and eukaryotes requires the recruitment and loading of a helicase at the replication origin. To subsequently unwind the double‐stranded DNA, the helicase must be properly positioned on the separated DNA strands. Several studies have revealed similarities and differences in the mechanisms used by different autonomously replicating DNA elements (replicons) for recruitment and activation of the appropriate helicase. Of particular interest are plasmid replicons that are adapted for replication in diverse bacterial hosts and are therefore intriguingly able to exploit the helicases of distantly related bacterial species. The different molecular mechanisms by which replicons recruit and load helicases are only just beginning to be understood.
NF‐κB activation by protein kinase C isoforms and B‐cell function
Authors: Moscat Jorge , Diaz‐Meco María T , Rennert Paul
Published: January 2003

B cells are essential to the immune response in health and disease. Results from knockout (KO) mice for different members of the nuclear factor‐κB (NF‐κB) family have highlighted the importance of this transcription factor in B cell development and function. The recent generation of additional KO mice for adapters and kinases implicated in NF‐κB activation, including several protein kinase C isoforms, has provided new insights into the roles of these proteins in B cell signalling. These studies have also given rise to a number of important questions that must be answered with further experimentation to establish accurately the signalling pathways that regulate B‐cell function through NF‐κB.
A rotary pumping model for helicase function of MCM proteins at a distance from replication forks
Authors: Laskey Ronald A , Madine Mark A
Published: January 2003

We propose an integrated model for eukaryotic DNA replication to explain the following problems: (1) How is DNA spooled through fixed sites of replication? (2) What and where are the helicases that unwind replicating DNA? (3) Why are the best candidates for replicative helicases, namely mini‐chromosome maintenance (MCM) proteins, not concentrated at the replication fork? (4) How do MCM proteins spread away from loading sites at origins of replication? We draw on recent discoveries to argue that the MCM hexameric ring is a rotary motor that pumps DNA along its helical axis by simple rotation, such that the movement resembles that of a threaded bolt through a nut, and we propose that MCM proteins act at a distance from the replication fork to unwind DNA. This model would place DNA replication in a growing list of processes, such as recombination and virus packaging, that are mediated by ring‐shaped ATPases pumping DNA by helical rotation.
Human cytomegalovirus prevents replication licensing by inhibiting MCM loading onto chromatin
Authors: Wiebusch Lüder , Uecker Ralf , Hagemeier Christian
Published: January 2003

To allow DNA replication only once per cell cycle, origins of replication are reactivated (‘licensed’) during each G1 phase. Licensing is facilitated by assembly of the pre‐replicative complex (pre‐RC) at origins that concludes with loading the mini‐chromosome maintenance (MCM) complex onto chromatin. Here we show that a virus exploits pre‐RC assembly to selectively inhibit cellular DNA replication. Infection of quiescent primary fibroblasts with human cytomegalovirus (HCMV) induces all pre‐RC factors. Although this is sufficient to assemble the MCM‐loading factors onto chromatin, as it is in serum‐stimulated cells, the virus inhibits loading of the MCM complex itself, thereby prematurely abrogating replication licensing. This provides a new level of control in pre‐RC assembly and a mechanistic rationale for the unusual HCMV‐induced G1 arrest that occurs despite the activation of the cyclin E‐dependent transcription programme. Thus, this particularly large virus might thereby secure the supply with essential replication factors but omit competitive cellular DNA replication.
The ligand‐sensitive gate of a potassium channel lies close to the selectivity filter
Authors: Proks Peter , Antcliff Jennifer F , Ashcroft Frances M
Published: January 2003

Potassium channels selectively conduct K+ ions across cell membranes and have key roles in cell excitability. Their opening and closing can be spontaneous or controlled by membrane voltage or ligand binding. We used Ba2+ as a probe to determine the location of the ligand‐sensitive gate in an inwardly rectifying K+ channel (Kir6.2). To a K+ channel, Ba2+ and K+ are of similar sizes, but Ba2+ blocks the pore by binding within the selectivity filter. We found that internal Ba2+ could still access its binding site when the channel was shut, which indicates that the ligand‐sensitive gate lies above the Ba2+‐block site, and thus within or above the selectivity filter. This is in marked contrast to the voltage‐dependent gate of KV channels, which is located at the intracellular mouth of the pore.
The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku
Authors: di Fagagna Fabrizio d'Adda , Weller Geoffrey R , Doherty Aidan J , Jackson Stephen P
Published: January 2003

Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA‐binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved.
Lymphotoxin and lipopolysaccharide induce NF‐κB‐p52 generation by a co‐translational mechanism
Authors: Mordmüller Benjamin , Krappmann Daniel , Esen Meral , Wegener Elmar , Scheidereit Claus
Published: January 2003

The ‘classical’ NF‐κB activation pathway proceeds via IκB kinase (IKK)‐β/γ‐mediated phosphorylation, induced ubiquitination and the degradation of small IκBs. An alternative, NF‐κB‐inducing kinase and IKK‐α‐dependent pathway, which stimulates the processing of NF‐κB2/p100, has recently been suggested. However, no physiological stimulus has been shown to trigger the activation of this pathway. Here we demonstrate that persistent stimulation with lymphotoxin β (LT‐β) receptor agonists or lipopolysaccharide (LPS), but not with interleukin‐1β, tumour necrosis factor‐α or 12‐O‐tetradecanoylphorbol‐13‐acetate, induces the generation of p52 DNA‐binding complexes by activating the processing of the p100 precursor. Induction of p52 DNA‐binding activity is delayed in comparison with p50/p65 complexes and depends on de novo protein synthesis. p100 is constitutively and inducibly polyubiquitinated, and both ubiquitination and p52 generation are coupled to continuing p100 translation. Thus, both LT‐β receptor agonists and LPS induce NF‐κB/p100 processing to p52 at the level of the ribosome.