bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of protein targets for mycophenolic acid acyl glucuronide in rat liver and colon tissue
Authors: Shipkova Maria , Beck Hermann , Voland Antje , Armstrong Victor W. , Gröne Hermann‐Josef , Oellerich Michael , Wieland Eberhard
Published: July 2004

Covalent binding of acyl glucuronides to proteins is considered an initiating event for the organ toxicity of drugs containing a carboxylic acid group. An acyl glucuronide (AcMPAG) of the immunosuppressant mycophenolic acid was described and shown to form covalent adducts with plasma albumin in vivo. The aim of the present investigation was to identify AcMPAG target proteins in the liver and colon of rats treated with mycophenolate mofetil, which may contribute to a better understanding of the mechanisms responsible for the development of side effects during therapy with this drug. Mycophenolate mofetil was administered per os in to Wistar rats (40 mg/kg/day) over 21 days. Proteins in liver and colon homogenates were separated by two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. AcMPAG labeled protein spots were detected by Western blotting. After in‐gel tryptic digestion of the protein spots from parallel gels (n = 2), peptides were characterized by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry. Data base searching identified AcMPAG target proteins. Tryptic peptides with sufficient signal intensities were subjected to post‐source decay analysis. Three proteins in the liver (ATPase/ATP synthase (alpha and beta subunits), protein disulfide isomerase A3 and selenium binding protein) and one protein in the colon (selenium binding protein) were identified as targets for AcMPAG. ATPase/ATP synthase and protein disulfide isomerase are essential proteins involved in the control of the energy and redox state of the cells, whereas the physiological role of selenium binding protein is not fully understood. This study shows for the first time the formation of adducts between tissue proteins and AcMPAG. Whether this chemical modification is associated with compromised protein function and drug toxicity remains to be investigated.
Profiling treatment‐specific post‐translational modifications in a complex proteome with subtractive substrate phage display
Authors: Tenzer Angela , Hofstetter Barbara , Sauser Christelle , Bodis Stephan , Schubiger August P. , Bonny Christophe , Pruschy Martin
Published: July 2004

Proteolytic activation of zymogens or controlled degradation of inhibitory factors is part of a major regulatory system on the post‐translational level to regulate treatment induced cellular stress responses. The identification of differential activity based substrates is thus of high interest to prioritize and validate candidate targets for drug discovery. Here we present a novel subtractive substrate phage display screening method for the selection of treatment induced post‐translational peptide modifications in complex proteomes. We investigated this approach with tumor cells in response to a protease activating anticancer treatment modality using subtractive and iterative screening of cellular extracts derived from control and treated cells. Specific phage were identified that served as substrates for proteolytic activities in response to treatment related activity changes and could be distinguished from substrates for unspecific proteolytic background activities. Novel, selected peptide substrates were investigated in vitro and in vivo and showed high substrate specificity and functional biological significance.
Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores
Authors: Huang Chun‐Ming , Foster K. Wade , DeSilva Tivanka S. , Van Kampen Kent R. , Elmets Craig A. , Tang De‐chu C.
Published: July 2004

The use of anthrax spores as a bioweapon has spurred efforts aimed at identifying key proteins expressed in Bacillus anthracis. Because spore germination and outgrowth occur prior to and are required for disease manifestations, blocking germination and early outgrowth with novel vaccines or inhibitors targeting critical B. anthracis germination and outgrowth‐associated factors is a promising strategy in mitigating bioterror. By screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores, respectively, we identified 10 spore proteins with statistically significant germination‐associated increases and decreases. It is likely that proteins whose levels change during germination may play key roles in the germination and outgrowth processes, and they should be listed as priority targets for development of prophylactic and therapeutic agents against anthrax. The 31 new proteins identified in this study also complement an emerging proteomic database of B. anthracis.
Protein composition of Paracoccus denitrificans cells grown on various electron acceptors and in the presence of azide
Authors: Bouchal Pavel , Přecechtělová Petra , Zdráhal Zbyněk , Kučera Igor
Published: July 2004

Two‐dimensional gel electrophoresis (2‐DE) with immobilized pH gradients was carried out on total cell lysates and membrane fractions of Paracoccus denitrificans with the aim to characterize differences in protein expression during growth under aerobic and various anaerobic conditions (with nitrate, nitrite or nitrous oxide). Comparative image analysis of the protein pattern revealed several subgroups of the total 800 protein spots resolved that were characteristically induced or repressed in response to individual electron acceptors. The respiratory inhibitor azide also exerted a profound influence upon cellular protein composition. However, since most of the proteins showing an altered expression pattern in cells growing on oxygen differed from those in cells growing on nitrite, we suppose that azide acts mainly indirectly, possibly by influencing other cellular signals. Limited information on the P. denitrificans genome has precluded the identification of more than eight protein spots as yet. A public accessible P. denitrificans 2‐DE protein database is currently built up at http://www.mpiib‐berlin.mpg.de/2D‐PAGE.
Incorporation of a new alkenyl‐based nonionic surfmer into acrylic latexes
Authors: Aramendia Esteban , Barandiaran María J. , De La Cal José C. , Grade Jo , Blease Trevor , Asua José M.
Published: July 2004

The mechanisms by which a new nonionic alkenyl‐based surfmer (Maxemul 5011™) was involved in acrylic emulsion polymerization were investigated. No proof of homopolymerization or of chain transfer to surfmer was obtained under the conditions studied. The effect of the initiator type, feeding time of the surfmer, particle size of the seed, and surfmer concentration, on kinetics and surfmer incorporation showed that the surfmer reacted mainly by copolymerization with the monomers in the outer shell of the polymer particles. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4202–4211, 2004
Synthesis of homopolymers and multiblock copolymers by the living ring‐opening metathesis polymerization of norbornenes containing acetyl‐protected carbohydrates with well‐defined ruthenium and molybdenum initiators
Authors: Miyamoto Yoshitaka , Fujiki Michiya , Nomura Kotohiro
Published: July 2004

The ring‐opening metathesis polymerization (ROMP) of norbornenes containing acetyl‐protected glucose [2,3,4,6‐tetra‐O‐acetyl‐glucos‐1‐O‐yl 5‐norbornene‐2‐carboxylate (1)] and maltose [2,3,6,2′,3′,4′,6′‐hepta‐O‐acetyl‐maltos‐1‐O‐yl 5‐norbornene‐2‐carboxylate (2)] was explored in the presence of Mo(N‐2,6‐iPr2C6H3)(CHCMe2Ph)(OtBu)2 (A), Ru(CHPh)(Cl)2(PCy3)2 (B; Cy = cyclohexyl), and Ru(CHPh)(Cl)2(IMesH2)(PCy3) (C; IMesH2 = 1,3‐dimesityl‐4,5‐dihydromidazol‐2‐ylidene). The polymerizations promoted by B and A proceeded in a living fashion with exclusive initiation efficiency, and the resultant polymers possessed number‐average molecular weights that were very close to those calculated on the basis of the monomer/initiator molar ratios and narrow molecular weight distributions (weight‐average molecular weight/number‐average molecular weight < 1.18) in all cases. The observed catalytic activity of B was strongly dependent on both the initial monomer concentration and the solvent employed, whereas the polymerization initiated with A was completed efficiently even at low initial monomer concentrations. The polymerization with C also took place efficiently, and even the polymerization with 1000 equiv of 1 was completed within 2 h. First‐order relationships between the propagation rates and the monomer concentrations were observed for all the polymerization runs, and the estimated rate constants at 25 °C increased in the following order: A > C > B. On the basis of these results, we concluded that ROMP with A was more suitable than ROMP with B or C for the efficient and precise preparation of polymers containing carbohydrates. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4248–4265, 2004
Orientation and relaxation of polymer–clay solutions studied by rheology and small‐angle neutron scattering
Authors: Malwitz Matthew M. , Butler Paul D. , Porcar Lionel , Angelette Drew P. , Schmidt Gudrun
Published: July 2004

The influence of shear on viscoelastic solutions of poly(ethylene oxide) (PEO) and clay [montmorillonite, i.e., Cloisite NA+ (CNA)] was investigated with rheology and small‐angle neutron scattering (SANS). The steady‐state viscosity and SANS were used to measure the shear‐induced orientation and relaxation of the polymer and clay platelets. Anisotropic scattering patterns developed at much lower shear rates than in pure clay solutions. The scattering anisotropy saturated at low shear rates, and the CNA clay platelets aligned with the flow, with the surface normal parallel to the gradient direction. The cessation of shear led to partial and slow randomization of the CNA platelets, whereas extremely fast relaxation was observed for laponite (LRD) platelets. These PEO–CNA networklike solutions were compared with previously reported PEO–LRD networks, and the differences and similarities, with respect to the shear orientation, relaxation, and polymer–clay interactions, were examined. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 3102–3112, 2004
Cell proliferation and apoptosis in prostate tumors and adjacent non‐malignant prostate tissue in patients at different time‐points after castration treatment
Authors: Ohlson Nina , Wikström Pernilla , Stattin Pär , Bergh Anders
Published: July 2004

Background

Androgen ablation is the standard treatment for advanced prostate cancer but the short‐term cellular effects are largely unknown.

Methods

Sextant prostate biopsies were taken from 77 prostate cancer patients before and 1–10 days after castration treatment. Apoptosis, cell proliferation, and morphology were studied in malignant and non‐malignant tissue, using stereological and immunohistochemical methods.

Results

Epithelial cell proliferation was significantly decreased both in non‐malignant and malignant epithelium already 1 day after therapy. It remained low until day 7, but increased thereafter in the remaining non‐malignant epithelial cells and in some tumors. Epithelial cell apoptosis was significantly increased during the first week and then returned to basal levels. The maximal apoptotic indexes, seven‐ and two‐times the intact levels in the non‐malignant and malignant glands, respectively, were found at days 3–4 or even earlier in the tumors. Signs of tumor shrinkage such as glandular collapse and decreased tumor cell size were observed from day 3 in most tumors.

Discussion

The present study shows that the magnitude and kinetics of the response to castration in the normal human prostate is very similar to the response previously described in rodents. We also demonstrate that most human prostate tumors rapidly respond to castration indicating the need for further evaluation of when and how to best monitor the effects of hormone ablation therapy in prostate cancer patients. © 2004 Wiley‐Liss, Inc.
Ability of PSA‐positive circulating macrophages to detect prostate cancer
Authors: Herwig Ralf , Horninger Wolfgang , Rehder Peter , Klocker Helmut , Ramoner Reinhold , Thurnher Martin , Pinggera Germar M. , Gozzi Christian , Konwalinka Günther , Bartsch Georg
Published: July 2004

Background

Prostate‐specific antigen (PSA) is a widely used serum marker for prostate cancer, but has a poor specificity for early detection at levels below 10 ng/ml, because it can also result from benign conditions. Our aim was to determine the frequencies of circulating PSA+ macrophages in a blinded study and to examine the suitability of this new method for differentiating between benign and malignant prostate disease.

Methods

Between October 2002 and February 2003, 126 patients undergoing transrectal biopsy were enrolled in this study. Peripheral blood macrophages were stained for intracellular content of PSA in all patients. Ten patients' peripheral blood mononulear cells (PBMCs) were also supplementarily stained for cytokeratin (CK) and epithelial membrane antigen (EMA). Macrophages were analysed by flow cytometry. Patients were grouped according to their biopsy histology and bone scan results.

Findings

Based on histological data, patients were classified as having no evidence of malignancy (NEM) (n = 59), prostatitis (n = 20), or localised prostate cancer (n = 37). Significantly higher levels of circulating PSA+ macrophages were found in prostate cancer compared to benign conditions. Calculating a 2% cut‐off level enabled the detection of localised prostate cancer with 89% sensitivity and 80% specificity. In a subset of patients (65%) with a serum PSA below 4 ng/ml and confirmed prostate cancer, the percentage of PSA+ macrophages was significantly higher compared to NEM and prostatitis. Macrophages of ten patients tested with prostate cancer contained significantly higher amounts of PSA, EMA, and CK compared to ten with NEM.

Interpretation

Intracellular PSA in combination with CK and EMA can be found in permeabilized blood macrophages, indicating phagocytosis of complete cancer cells. This study further suggests, that this new method might be suitable for differentiating between prostate cancer and benign conditions especially in patients with low serum PSA. © 2004 Wiley‐Liss, Inc.
Monitoring of skeletal progression of prostate cancer by GFP imaging, X‐ray, and serum OPG and PTHrP
Authors: Burton D.W. , Geller J. , Yang M. , Jiang P. , Barken I. , Hastings R.H. , Hoffman R.M. , Deftos L.J.
Published: July 2004

Background

Prostate cancers (PCas) produce factors that can serve as biomarkers for tumor metastasis and bone progression. Transduced GFP expression by cancer cells can be imaged to monitor therapy. We exploited both concepts by developing a GFP‐expressing PCa cell line that expresses PTHrP and studying it in an animal model of malignancy with methods that assess the skeletal progression of this tumor.

Methods

We developed a GFP‐producing PCa cell line by stable transduction of PC‐3 PCa cells. This PC‐3 variant was used to study tumor progression in an immunocompromised mouse model. Skeletal progression of the PCa cells and the effects of pamidronate administration were evaluated radiologically, fluorometrically, and by measurement of serum tumor markers.

Results

The PC‐3 cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC‐3 cells could be monitored by GFP optical imaging, X‐ray, and by measurements of tumor products in serum, notably PTHrP and OPG. Pamidronate treatment reduced tumor burden as assessed at autopsy by imaging and biomarkers.

Conclusions

Pamidronate treatment exhibited anti‐tumor effects that were reflected by decreases in serum PTHrP, OPG, and by GFP and radiological imaging procedures. Imaging of GFP expression enables real‐time monitoring of tumor growth in the bone. PTHrP and OPG may be useful as tumor biomarkers for PCa that has metastasized to bone. This novel human PCa model can be used to study the clinical potential of diagnostic and therapeutic modalities in the skeletal progression of PCas. © 2004 Wiley‐Liss, Inc.
Castration triggers growth of previously static androgen‐independent lesions in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model
Authors: Johnson Mac A. , Iversen Philip , Schwier Phillip , Corn Angela L. , Sandusky George , Graff Jeremy , Neubauer Blake Lee
Published: July 2004

Background

Androgen‐deprivation remains the standard of care for metastatic prostate cancer, yet its total impact on the course of disease is incompletely established.

Methods

We have examined the long‐term effects of castration upon the progression of established cancer in the TRAMP transgenic mouse model of prostate cancer. Mice castrated at 15‐weeks of age, as well as intact littermates, were followed until spontaneous death from cancer.

Results

Statistical analyses of age‐at‐death versus primary tumor mass revealed that mice segregate into two categories of response to androgen‐deprivation. In Category One, the act of castration paradoxically triggers the growth of microscopic androgen‐independent lesions, as evidenced by a statistical synchronization of primary tumor growth. Delaying castration until 20‐weeks of age delays the synchronized growth of these tumors, as well as the deaths of the host mice. In Category Two, castration eliminates or substantially delays primary tumor growth, but fails to eliminate metastases. so that castration is found to impart no long‐term survival advantage.

Conclusions

We propose a two‐step model for the alteration of androgen signaling in prostate cancer capable of explaining the above paradoxical results, which is based upon the dualistic role of androgens as both survival and differentiation factors. This model makes specific predictions for clinical intervention that are discussed in light of published studies. © 2004 Wiley‐Liss, Inc.
Prostate specific membrane antigen (PSMA) is a tissue‐specific target for adenoviral transduction of prostate cancer in vitro
Authors: Kraaij R. , van Rijswijk A.L.C.T. , Oomen M.H.A. , Haisma H.J. , Bangma C.H.
Published: July 2004

Background

Adenovirus binds to the coxsackievirus and adenovirus receptor (CAR) as a first step in the process of cellular infection. This dependence on CAR potentially limits the use of adenovirus in gene therapy, since CAR is expressed in many tissues of the body, and expression of CAR may be low or lost upon progression of certain tumors. These limitations may be overcome by transductional targeting of adenovirus towards other cell surface molecules. We have evaluated the pantumoral epithelial cell adhesion molecule (EpCAM) and prostate specific membrane antigen (PSMA) as possible targets for adenoviral transduction of prostate cancer cells.

Methods

Bispecific antibodies, constructed as conjugates between an anti‐adenovirus fiber knob Fab' fragment and anti‐EpCAM or anti‐PSMA monoclonal antibodies, were incubated with an eGFP‐expressing adenovirus to retarget this vector. A cell panel, that includes two prostate cancer cell lines and four non‐prostate control lines, were infected with serial dilutions of the retargeted vector and specificity of infection was determined.

Results

Receptor‐specific transduction was obtained for both EpCAM and PSMA. PSMA‐retargeting was shown to be selective for the prostate cancer cell lines.

Conclusions

PSMA serves as a tissue‐specific target for adenoviral vectors and may be applicable for gene therapeutical treatment of prostate cancer. © 2004 Wiley‐Liss, Inc.
Theoretical study of the stability of wurtzite, zinc‐blende, NaCl and CsCl phases in group IIIB and IIIA nitrides
Authors: Mancera Luis , Rodríguez Jairo A. , Takeuchi Noboru
Published: July 2004

The relative stability of wurtzite, zinc‐blende, rock‐salt and CsCl structures and the possibility of high‐pressure phase transformations from wurtzite to rocksalt and from rocksalt to CsCl is of special interest. We present a comparative study of the stability of these phases for two Group IIIB‐nitrides (ScN, YN) and two Group IIIA‐nitrides (GaN, InN), in the framework of DFT. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Calibration of a pH sensitive buried channel silicon‐on‐insulator MOSFET for sensor applications
Authors: Ashcroft B. , Takulapalli B. , Yang J. , Laws G. M. , Zhang H. Q. , Tao N. J. , Lindsay S. , Gust D. , Thornton T. J.
Published: July 2004

The threshold voltage characteristics of a buried channel silicon‐on‐insulator MOSFET is examined in solutions of varying acidity. Experiments utilizing an integrated micro‐fluidic channel exhibit a variation in threshold voltage that appears approximately linear with pH in the range from pH 4 to pH 7, with a sensitivity of ∼1 V per pH unit. Charge configuration changes in the vicinity of the MOSFET inversion layer due to protonation/deprotonation of the device surface is proposed as an explanation for the observed shifts in threshold voltage. When the pH range is expanded we observe a non‐linear relationship bet‐ ween pH and the threshold voltage of the device, this behavior is explained in terms of deprotonation of the different species of the native oxide surface, Numerical simulations of the MOSFET demonstrate that the threshold voltage sensitivity corresponds to an additional surface positive fixed charge density of ∼ 1 × 1010 cm–2 for each pH unit. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Metabolism of 1‐{3‐[3‐(4‐cyanobenzyl)‐3H‐imidazol‐4‐yl]‐propyl}‐3‐(6‐methoxypyridin‐3‐yl)‐1‐(2‐trifluoromethylbenzyl)thiourea (YH3945), a novel anti‐cancer drug, in rats using the 14C‐labeled compound
Authors: Lee Jaeick , La Sookie , Ahn Byung Rak , Jeong Tae Cheon , Kim Dong‐Hyun
Published: July 2004

The metabolism of a novel anti‐cancer agent, 1‐{3‐[3‐(4‐cyanobenzyl)‐3H‐imidazol‐4‐yl]‐propyl}‐3‐(6‐methoxypyridin‐3‐yl)‐1‐(2‐trifluoromethylbenzyl)thiourea (YH3945), was investigated in rats. Bile, plasma, feces, and urine were collected and analyzed by a high‐performance liquid chromatography (HPLC) system equipped with ultraviolet (UV), mass spectrometric, and radioactivity detectors. After intravenous dosing, mean radiocarbon recovery was 74.4 ± 1.3% with 62.4 ± 1.2% in the feces and 12.0 ± 0.5% in the urine. Biliary excretion of the radioactivity for the first 24 h period was approximately 32%, suggesting that YH3945 is cleared by hepatobiliary excretion. YH3945 was extensively metabolized to 21 different metabolites including glucuronide conjugates, and structures of the metabolites were elucidated based on MSn and NMR spectral analyses. The major metabolic pathways in the rat were identified as O‐demethylation of methoxypyridine, N‐debenzylation of imidazole, and hydroxylation. Cyclic metabolites were also identified; concomitant demethylation in the methoxypyridine moiety and hydroxylation at the C16 position might destroy the chemical stability of the compound and subsequently lead to non‐enzymatic cyclization. Cyclic metabolites were characteristic of YH3945, and a non‐enzymatic reaction mechanism for the formation of cyclic metabolites was postulated. Copyright © 2004 John Wiley & Sons, Ltd.
Analysis of neutral drugs in human plasma by fluoride attachment in liquid chromatography/negative ion electrospray tandem mass spectrometry
Authors: Sheen J. F. , Her G. R.
Published: July 2004

The analysis of several neutral drugs, mephenesin, guaifenesin, simvastatin, podophyllotoxin and inositol, was accomplished by negative ion electrospray ionization mass spectrometry (ESI‐MS) using adduct formation with three different halide ions. The fluoride, chloride and bromide adducts of the selected drugs exhibited intense signals in negative ion ESI. Under collision‐induced dissociation, the major product ions of bromide and chloride adducts were the nonspecific bromide and chloride anions, respectively. In contrast, fluoride adducts produced strong [MH]− ions as well as product ions with good intensity. Fluoride attachment liquid chromatography/negative ion electrospray tandem mass spectrometry (LC/ESI‐MS/MS) was applied to the analysis of the selected neutral drugs in human plasma. Detection limits in the range of 0.025–0.05 ng/mL were achieved using 0.5 mL plasma. Good linearity was observed for each of the drugs examined in human plasma over the range of 0.05–50 ng/mL. Copyright © 2004 John Wiley & Sons, Ltd.
Proteome analysis in the bovine adrenal medulla using liquid chromatography with tandem mass spectrometry
Authors: Jalili Pegah R. , Dass Chhabil
Published: July 2004

Liquid chromatography/mass spectrometry (LC/MS)‐based proteomics has been used to identify soluble proteins in the bovine adrenal medulla. This gland is a major source of hormones, opioids, neurotransmitters, and several vital proteins. The adrenal medulla proteins were first purified using ammonium sulfate precipitation. The resulting proteins were then pre‐fractionated with a C‐4 high‐peformance liquid chromatography (HPLC) column. Each 2‐min HPLC fraction was digested with trypsin, and separated further and analyzed using capillary liquid chromatography/tandem mass spectrometry (capLC/nanospray‐MS/MS) to map the proteome of the adrenal medulla. The parent mass and sequence ion information thus obtained for tryptic peptides was used to search the NCBInr database using the SEQUEST search engine. A total of 195 proteins were identified, of which 71 had good scores (delta correlation value greater than 0.1, preliminary score above 200, and cross‐correlation value above 2.5). The prominent proteins thus identified are secretogranin I precursor, chromogranin A, proenkephalin A precursor, myosin X, hemoglobin beta chain, hemoglobin alpha chain, heat shock protein 10 kDa, and replicase. Copyright © 2004 John Wiley & Sons, Ltd.
Controlling matrix suppression for matrix‐assisted laser desorption/ionization analysis of small molecules
Authors: Donegan Michael , Tomlinson Andy J. , Nair Hari , Juhasz Peter
Published: July 2004

The need for high‐throughput methodologies providing both qualitative and quantitative information has grown substantially in the pharmaceutical laboratory in recent years. Currently, tandem mass spectrometry (MS/MS) using quadrupole technology offers analysis in the minutes time scale. The use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) offers the advantage of speed and automation and enables analysis in the seconds time scale with accurate mass capabilities that are not typically found in quadrupole MS/MS. However, one of the limitations of MALDI for the analysis of small molecules is the abundance of interfering matrix peaks in the low molecular weight region of the mass spectrum. Described herein is an evaluation of a pre‐prepared MALDI target plate that has been coated with a thin layer of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and nitrocellulose. This modified plate has been shown to suppress or eliminate CHCA matrix signals without any significant loss of analyte sensitivity when compared with analysis of the same sample using an unmodified target plate. Copyright © 2004 John Wiley & Sons, Ltd.
Investigation of non‐covalent interactions between paramagnetic complexes and human serum albumin by electrospray mass spectrometry
Authors: Henrotte Virginie , Laurent Sophie , Gabelica Valérie , Elst Luce Vander , Depauw Edwin , Muller Robert N.
Published: July 2004

Stable gadolinium(III) chelates are nowadays routinely used as contrast agents for magnetic resonance imaging (MRI). Their non‐covalent binding to human serum albumin (HSA) has shown to improve their efficacy. Non‐covalent interactions lead to complex formation that can be quantified by several techniques that are mostly tedious and time‐consuming. In this study, electrospray ionization mass spectrometry (ESI‐MS) was used to investigate the interaction between HSA and several gadolinium(III) complexes. The results were compared with those obtained in the liquid phase. Four gadolinium complexes were investigated: Gd‐DTPA 1, Gd‐C4Me‐DTPA 2, Gd‐EOB‐DTPA 3, and MP‐2269 4. Relaxometry studies show that complexes 1 and 2 have no significant affinity for HSA, while complexes 3 and 4 have increasing affinities for the protein. 1:1 and 1:2 complexes between HSA and MP‐2269 were detected by ESI‐MS for a twofold excess of the contrast agent, whereas a ligand/protein molar ratio of 4:1 was necessary to observe a 1:1 stoichiometry for Gd‐EOB‐DTPA, an observation that is in good agreement with the known weaker affinity of the contrast agent for the protein. At a fourfold molar excess, no supramolecular complex was observed for Gd‐DTPA 1 and Gd‐C4Me‐DTPA 2; a tenfold molar excess was necessary to detect a 1:1 complex, confirming the very weak affinity of these contrast agents for HSA. Copyright © 2004 John Wiley & Sons, Ltd.
Chronic immobilization stress differentially affects the expression of BDNF mRNA and protein in the mouse hippocampus
Authors: Adlard Paul A. , Perreau Victoria M. , Cotman Carl W.
Published: July 2004

Stress impacts multiple systems within the CNS, including the hippocampus. A key molecular mediator in hippocampal functions, including learning and memory, is brain‐derived neurotrophic factor (BDNF). BDNF mRNA is reported to be decreased following chronic (every day) immobilization stress (CIS) in the rat, however the effect on protein is unknown. Utilizing a mouse model of CIS, the hypothesis that total hippocampal BDNF protein, in addition to BDNF mRNA, would be decreased was tested. The effects of intermittent (alternating day) immobilization and cold stress on hippocampal BDNF protein levels was also examined.

In this study it was demonstrated that CIS results in a small (8 per cent decrease relative to non‐stressed controls), but significant (p = 0.0013) decrease in the exon coding for mature BDNF (exon VI). In addition, there was a significant (34 per cent, p = 0.005) decrease in the mRNA coding for exon I in the stressed animals. Contrary to expectations, however, CIS resulted in a significant (116 per cent, p = 0.0087) increase in hippocampal BDNF protein levels. Furthermore, both intermittent immobilization and chronic cold stress both increased BDNF protein levels. Chronic stress, therefore, differentially regulates BDNF mRNA and protein levels in the hippocampus. Copyright © 2004 John Wiley & Sons, Ltd.
A cultural perspective of the induction of five reform‐minded beginning mathematics and science teachers
Authors: McGinnis J. Randy , Parker Carolyn , Graeber Anna O.
Published: July 2004

The purpose of this empirical study was to present a detailed description and interpretation of what happens in schools to beginning teachers who are prepared to enact reform‐based practices in mathematics and science. The focus was on a select sample of graduates from the Maryland Collaborative for Teacher Preparation [MCTP], a statewide reform‐based undergraduate teacher preparation program funded by the National Science Foundation. Interpretative research methodology was used to conduct a cultural case study of the beginning teachers' first 2 years of practice (first year, N = 5; second year, N = 3). We documented differential experiences and perceptions of the beginning teachers from both inside (emic) and outside (etic) perspectives. Documented discussion centered on an analytical framework suggested elsewhere. Findings were framed in two components: the individual's intentions, needs, and capabilities; and the institutional demands, affordances, and constraints. The major insight was that the beginning teachers' perception of their school culture was a major factor in whether reform‐aligned mathematics and science teaching was regularly implemented by the beginning teachers. In instances where the beginning teachers' perceived that their school cultures offered a lack of support for their intent to implement reform‐based practices the beginning teachers exhibited differing social strategies (resistance, moving on, and exit). Therefore, to sustain reform (and, by extension, to retain beginning mathematics and science teachers), a key implication is to place additional attention on the use of the school culture perspective to improve teacher preparation and induction experiences. © 2004 Wiley Periodicals, Inc. J Res Sci Teach 41: 720–747, 2004
Young children's emotional practices while engaged in long‐term science investigation
Authors: Zembylas Michalinos
Published: July 2004

In this article, the role of young children's emotional practices in science learning is described and analyzed. From the standpoint of performativity theory and social‐constructionist theory of emotion, it is argued that emotion is performative and the expression of emotion in the classroom has its basis in social relationships. Arising from these relationships is the emotional culture of the classroom that plays a key role in the development of classroom emotional rules as well as the legitimation of science knowledge. These relationships are reflected in two levels of classroom dialogue: talking about and doing science, and expressing emotions about science and its learning. The dynamics of the negotiations of classroom emotional rules and science knowledge legitimation may dispose students to act positively or negatively toward science learning. This analysis is illustrated in the experiences of a teacher and her students during a 3‐year ethnographic study of emotions in science teaching and learning. This research suggests the importance of the interrelationship between emotions and science learning and the notion that emotional practices can be powerful in nurturing effective and exciting science learning environments. © 2004 Wiley Periodicals, Inc. J Res Sci Teach 41: 693–719, 2004
Efficient gene targeting in Kluyveromyces lactis
Authors: Kooistra Rolf , Hooykaas Paul J. J. , Steensma H. Yde
Published: July 2004

Integration of a DNA fragment in a host genome requires the action of a double‐strand break (DSB) repair mechanism. Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non‐homologous end joining (NHEJ). In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency. To study and to improve gene targeting efficiency, K. lactis has been used as a model. The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed. Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences. In wild‐type K. lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks. The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks. These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was achieved by the transformation of wild‐type K. lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments. Using this method, PCR‐generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement. Copyright © 2004 John Wiley & Sons, Ltd.
Localization of proteins that are coordinately expressed with Cln2 during the cell cycle
Authors: Sundin Bryan A. , Chiu Chun‐Hwei , Riffle Michael , Davis Trisha N. , Muller Eric G. D.
Published: July 2004

The localization of proteins can give important clues about their function and help sort data from large‐scale proteomic screens. Forty‐five proteins were tagged with the GFP variant YFP. These proteins were chosen because they are encoded by genes that display strong cell cycle‐dependent expression that peaks in G1. Most of these proteins localize to either the nucleus or to sites of cell growth. We are able to assign new cellular component GO terms to ASF2, TOS4, RTT109, YBR070C, YKR090W, YOL007C, YOL019W and YPR174C. We also have localization data for 21 other proteins. Noteworthy localizations were found for Rfa1p, a member of the DNA replication A complex, and Pri2p and Pol12p, subunits of the α‐DNA polymerase : primase complex. In addition to its nuclear localization, Rfa1p assembled into cytoplasmic foci adjacent to the nucleus in cells during the G1–S phase transition of the cell cycle. Pri2 and Pol12 took on a beaded appearance at the G1–S transition and later in the cell cycle were enriched in the nuclear envelope. A new spindle pole body/nuclear envelope component encoded by YPR174 was identified. The cell cycle‐dependent abundance of Tos4p mirrored Yox1p and these two proteins were the only proteins that were found exclusively at the G1–S phase of the cell cycle. A complete list of localizations, along with images, can be found at our website (http://www.yeastrc.org/cln2/). Copyright © 2004 John Wiley & Sons, Ltd.
Phenotypic characterization of glucose repression mutants of Saccharomyces cerevisiae using experiments with 13C‐labelled glucose
Authors: Raghevendran Vijayendran , Gombert Andreas Karoly , Christensen Bjarke , Kötter Peter , Nielsen Jens
Published: July 2004

In the field of metabolic engineering and functional genomics, methods for analysis of metabolic fluxes in the cell are attractive as they give an overview of the phenotypic response of the cells at the level of the active metabolic network. This is unlike several other high‐throughput experimental techniques, which do not provide information about the integrated response a specific genetic modification has on the cellular function. In this study we have performed phenotypic characterization of several mutants of the yeast Saccharomyces cerevisiae through the use of experiments with 13C‐labelled glucose. Through GC–MS analysis of the 13C incorporated into the amino acids of cellular proteins, it was possible to obtain quantitative information on the function of the central carbon metabolism in the different mutants. Traditionally, such labelling data have been used to quantify metabolic fluxes through the use of a suitable mathematical model, but here we show that the raw labelling data may also be used directly for phenotypic characterization of different mutant strains. Different glucose derepressed strains investigated employed are the disruption mutants reg1, hxk2, grr1, mig1 and mig1mig2 and the reference strain CEN.PK113‐7D. Principal components analysis of the summed fractional labelling data show that deleting the genes HXK2 and GRR1 results in similar phenotype at the fluxome level, with a partial alleviation of glucose repression on the respiratory metabolism. Furthermore, deletion of the genes MIG1, MIG1/MIG2 and REG1 did not result in a significant change in the phenotype at the fluxome level. Copyright © 2004 John Wiley & Sons, Ltd.
Differential expression and localization of CFTR and ENaC in mouse endometrium during pre‐implantation
Authors: Yang Jian Zhi , Ajonuma Louis Chukwuemeka , Tsang Lai Ling , Lam Sun Yee , Rowlands Dewi Kenneth , Ho Lok Sze , Zhou Chen Xi , Chung Yiu Wa , Chan Hsiao Chang
Published: July 2004

Interaction between the cystic fibrosis transmembrane conductance regulator (CFTR), a CAMP‐activated Cl− channel, and epithelial Na+ channel (ENaC) has been proposed as the major mechanism regulating uterine fluid absorption and secretion. Differential expression of these ion channels may give rise to dynamic changes in the fluid environment affecting various reproductive events in the female reproductive tract. This study investigated the expression and localization of CFTR and ENaC during the pre‐implantation period. Semi‐quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry were used to study the expression and localization of CFTR and ENaC in uteri collected from mature superovulated female mice. RT‐PCR showed maximal ENaC and CFTR expression on day 3 after mating. Maximal immunoreactivity was also observed for both ENaC and CFTR on day 3 after mating. However, ENaC was immunolocalized to the apical membrane of both luminal and glandular epithelia, while CFTR was predominantly found in the stromal cells rather than the epithelial cells. Differential expression and localization of CFTR and ENaC provide a molecular mechanism by which maximal fluid absorption can be achieved immediately prior to implantation, to ensure the immobilization of the blastocyst necessary for implantation.
Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain
Authors: Oliveira Maria Cláudia G. , Parada Carlos Amílcar , Veiga Maria Cecília Ferraz Arruda , Rodrigues Luciane Rocha , Barros Silvana Pereira , Tambeli Cláudia Herrera
Published: July 2004

Evidence is accumulating which supports a role for ATP in the initiation of pain by acting on P2X receptors expressed on nociceptive afferent nerve terminals. To investigate whether these receptors play a role in temporomandibular (TMJ) pain, we studied the presence of functional P2X receptors in rat TMJ by examining the nociceptive behavioral response to the application of the selective P2X receptor agonist α,β‐methylene ATP (α,β‐meATP) into the TMJ region of rat. The involvement of endogenous ATP in the development of TMJ inflammatory hyperalgesia was also determined by evaluating the effect of the general P2 receptor antagonist pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on carrageenan‐induced TMJ inflammatory hyperalgesia. Application of α,β‐meATP into the TMJ region of rats produced significant nociceptive responses that were significantly reduced by the co‐application of lidocaine N‐ethyl bromide quaternary salt, QX‐314, (2%) or of the P2 receptor antagonist PPADS. Co‐application of PPADS with carrageenan into the TMJ significantly reduced inflammatory hyperalgesia. The results indicate that functional P2X receptors are present in the TMJ and suggest that endogenous ATP may play a role in TMJ inflammatory pain mechanisms possibly by acting primarily in these receptors.
Paraneoplastic leukocytosis with mature neutrophilia in a cat with pulmonary squamous cell carcinoma
Authors: Dole Robert S. , MacPhail Catriona M. , Lappin Michael R.
Published: July 2004

A 13-year old cat had 3-day duration of vomiting, lethargy, and anorexia. A complete blood count revealed a severe neutrophilia (126.9×103 cellsμl). Thoracic radiographs demonstrated a large solitary lung mass. A bone marrow aspirate documented myeloid hyperplasia. A left intercostal thoracotomy was performed and left cranial and caudal lung lobectomies were performed in order to remove the mass in its entirety. Histological diagnosis was squamous cell carcinoma of the lung. Following surgery, the severe neutrophilia began to decrease. It was in the normal range approximately 6 weeks postoperatively. The presence of a primary lung tumor combined with a mature neutrophilia with resolution following surgical resection suggests a paraneoplastic syndrome.
Parvovirus vectors: use and optimisation in cancer gene therapy
Authors: Blechacz Boris , Russell Stephen J.
Published: July 2004

With the increasing incidence and mortality of cancer worldwide, there is an urgent need for new therapeutic approaches. Gene therapy is one such approach and preliminary data are promising. Viral and nonviral vector systems for gene delivery are available, but most of the current systems suffer from disadvantages such as low transfection efficiencies, in vivo instability, targeting problems, mutagenic potential and immunogenicity. Viruses of the Parvoviridae family, which are characterised by their oncotropism, oncosuppression, long-term gene expression and human apathogenicity, potentially offer advantages as viral vectors. This article evaluates their usefulness in gene therapy strategies for cancer.
Three‐dimensional, multispecies, high spatial resolution MHD studies of the solar wind interaction with Mars
Authors: Ma Yingjuan , Nagy Andrew F. , Sokolov Igor V. , Hansen Kenneth C.
Published: July 2004

We present the results of model calculations, using our new, four‐species, spherical MHD model. Our results are compared with the relevant and limited available data. The resulting comparisons help us to increase our understanding of the interaction processes between the solar wind and the Martian atmosphere/ionosphere. This new model with a spherical grid structure allowed us to use small (∼10 km) radial grid spacing in the ionospheric region. We found that the calculated bow shock positions agree reasonably well with the observed values. The calculated results vary with interplanetary magnetic field orientation, solar cycle conditions, and subsolar location. We found that our calculated ion densities, with parameters corresponding to solar cycle minimum conditions, reproduced the Viking 1 observed ion densities well. The calculated solar cycle maximum densities, above ∼140 km, are also consistent with the appropriate Mars Global Surveyor radio occultation electron densities. Both the calculated solar cycle maximum and solar cycle minimum total transterminator and escape fluxes are significantly smaller than our previously published values. This decrease is due to the improved temperature values used for the recombination rates in this new model, which in turn results in lower ion densities and lower fluxes.
A technology‐based global inventory of black and organic carbon emissions from combustion
Authors: Bond Tami C. , Streets David G. , Yarber Kristen F. , Nelson Sibyl M. , Woo Jung‐Hun , Klimont Zbigniew
Published: July 2004

We present a global tabulation of black carbon (BC) and primary organic carbon (OC) particles emitted from combustion. We include emissions from fossil fuels, biofuels, open biomass burning, and burning of urban waste. Previous “bottom‐up” inventories of black and organic carbon have assigned emission factors on the basis of fuel type and economic sector alone. Because emission rates are highly dependent on combustion practice, we consider combinations of fuel, combustion type, and emission controls and their prevalence on a regional basis. Central estimates of global annual emissions are 8.0 Tg for black carbon and 33.9 Tg for organic carbon. These estimates are lower than previously published estimates by 25–35%. The present inventory is based on 1996 fuel‐use data, updating previous estimates that have relied on consumption data from 1984. An offset between decreased emission factors and increased energy use since the base year of the previous inventory prevents the difference between this work and previous inventories from being greater. The contributions of fossil fuel, biofuel, and open burning are estimated as 38%, 20%, and 42%, respectively, for BC, and 7%, 19%, and 74%, respectively, for OC. We present a bottom‐up estimate of uncertainties in source strength by combining uncertainties in particulate matter emission factors, emission characterization, and fuel use. The total uncertainties are about a factor of 2, with uncertainty ranges of 4.3–22 Tg/yr for BC and 17–77 Tg/yr for OC. Low‐technology combustion contributes greatly to both the emissions and the uncertainties. Advances in emission characterization for small residential, industrial, and mobile sources and top‐down analysis combining field measurements and transport modeling with iterative inventory development will be required to reduce the uncertainties further.