bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of MitoTracker Green™ and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues
Authors: Pendergrass W. , Wolf N. , Poot M.
Published: August 2004

Background

Chloromethyl‐X‐rosamine (CMXRos) and MitoTracker Green (MTG) have proved to be useful dyes with which to measure mitochondrial function. CMXRos is a lipophilic cationic fluorescent dye that is concentrated inside mitochondria by their negative mitochondrial membrane potential (MMP). MTG fluorescence has been used as a measure of mitochondrial mass independent of MMP. The fluorescence ratio of the two dyes is a relative measure of the MMP independent of mitochondrial mass. Because MTG was recently reported to be sensitive to MMP, we have reevaluated the effects of loss of MMP on MTG and CMXRos fluorescence, using both flow cytometry and laser scanning confocal microscopy (LSCM).

Methods

Using flow cytometry, the relative fluorescence of CMXRos, R123, and MTG was determined in human lymphoblastoid cell lines (LCLs) with or without carbonyl cyanide p‐trifluoromethoxylphenyl‐hydrazone (FCCP), used to collapse the MMP. LSCM analysis was also used to evaluate the effect of FCCP on MTG and CMXRos fluorescence of mouse cells and viable lenses in culture. The cytotoxicity of the dyes was determined using flow analysis of endogenous NADH fluorescence. The sensitivity of MTG fluorescence to H2O2 was also evaluated using flow cytometry.

Results

CMXRos fluorescence was dependent on MMP, whereas MTG fluorescence was not affected by MMP, using either flow or LSCM. Specific staining of mitochondria was seen with both dyes in all cell types tested, without evidence of cytotoxicity, as determined by NADH levels. H2O2 damage slightly increased MTG staining of cells.

Conclusions

Our results indicate that CMXRos is a nontoxic sensitive indicator of relative changes in MMP, whereas MTG is relatively insensitive to MMP and oxidative stress, using both flow and LSCM analyses, provided optimal staining conditions are used. In addition, these dyes can be useful for the study of mitochondrial morphology and function in whole tissues, using LSCM. © 2004 Wiley‐Liss, Inc.
Characterization of cell‐cycle progression and growth of WB‐F344 normal rat liver epithelial cells following gamma‐ray exposure
Authors: Gerashchenko Bogdan I. , Azzam Edouard I. , Howell Roger W.
Published: August 2004

Background

Apparently normal rat liver epithelial cells (WB‐F344) have been widely used in studies pertaining to carcinogenesis. Ionizing radiation, a well known carcinogen, is known to perturb cell‐cycle progression in a dose‐dependent manner, thereby causing delay in cell proliferation. However, for WB‐F344 cells, there is a paucity of such data, which are of substantial importance in understanding their radiation response. Here, the distribution of phases in the cell‐cycle and the proliferation ability of WB‐F344 cells are characterized at various time points after the cells have been irradiated with different doses of γ‐rays.

Methods

After WB‐F344 cells reached 100% confluence, they were trypsinized and suspended at 3.5 × 105 cells/ml in culture medium. Cells were irradiated in suspension with 137Cs γ‐rays at doses from 1–10 Gy. After irradiation, 1 × 105 cells were plated into 60 × 15‐mm culture dishes and incubated at 37°C, with 2% CO2 and 98% air. At 12, 24, 36, 48, and 60 h postirradiation, cells were harvested, counted, and subjected to flow cytometric cell‐cycle analysis.

Results

Growth curves of WB‐F344 cells irradiated with γ‐rays started to separate at 36 h postirradiation. By 60 h postirradiation, the growth curves for each of the 10 absorbed doses were distinctly separated. Drastic redistributions of control and irradiated cells within G0/G1‐, S‐, and G2/M‐phases of the cell cycle were observed during the first 36 h of cell growth. At each time point postirradiation, cell‐cycle phase profiles of irradiated cells were altered in a dose‐dependent manner. In general, there was a strong correlation between the percentage of G2/M‐phase cells and absorbed dose, with the exception of 24 h postirradiation. The percentage of G2/M‐phase cells increased as a function of time postirradiation, suggestive of delays in the passage of cells through the G2 cell‐cycle checkpoint.

Conclusions

This work provides a general description of cell cycle redistribution and repopulation kinetics of WB‐F344 cells at various times postirradiation of quiescent cells that were subsequently allowed to proliferate. In general, growth inhibition and delays in progression through G2/M‐phase correlated well with radiation dose. These data should be of considerable significance in the design of experiments that examine the radiation response of these cells. © 2004 Wiley‐Liss, Inc.
High‐throughput laser‐mediated in situ cell purification with high purity and yield
Authors: Koller Manfred R. , Hanania Elie G. , Stevens Janine , Eisfeld Timothy M. , Sasaki Glenn C. , Fieck Annabeth , Palsson Bernhard Ø.
Published: August 2004

Background

Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield.

Methods

An automated closed‐loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser‐based semiconductor manufacturing equipment. Laser‐enabled analysis and processing has combined high‐throughput in situ cell imaging with laser‐mediated cell manipulation via large field‐of‐view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility.

Results

Photothermal purification required approximately 108 W/cm2 at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 109 W/cm2 at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 1010 W/cm2 independent of wavelength, resulting in immediate cell lysis. Each approach resulted in high efficiency purification (>99%) after a single operation, as demonstrated with eight cell types. An automated closed‐loop process to re‐image and irradiate remaining targets in situ was implemented, resulting in improved purification (99.5–100%) without decreasing cell yield or affecting sterility in this closed system. Efficient purification was demonstrated with B‐ and T‐cell mixtures over a wide range of contaminating cell percentages (0.1–99%) and cell densities (104‐106/cm2). Efficient cloning of 293T cells based on fluorescence with green fluorescent protein after plasmid transfection was also demonstrated.

Conclusions

In situ laser‐mediated purification was achieved with nonadherent and adherent cells on the automated laser‐enabled analysis and processing platform. Closed‐loop processing routinely enabled greater than 99.5% purity with a greater than 90% cell yield in sample sizes ranging from 101 to 108 cells. Throughput ranged from approximately 103 to 105 total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively. © 2004 Wiley‐Liss, Inc.
Exercise as a therapeutic intervention for the prevention and treatment of insulin resistance
Authors: Hawley John A.
Published: August 2004

During the past half‐century, there has been a dramatic increase in the incidence of obesity, insulin resistance and type 2 diabetes. A hallmark feature of these conditions is impaired glucose tolerance (IGT). Cross‐sectional and retrospective epidemiological studies have provided direct evidence that a lack of physical activity is strongly associated with IGT. Indeed, physical inactivity is an independent risk factor for insulin resistance and type 2 diabetes. The primary defect(s) in the development of whole‐body insulin resistance remain unclear. However, during the past decade significant progress has been made towards an understanding of the molecular basis underlying the beneficial effects of exercise training in stimulating the entry of glucose into insulin‐sensitive tissues. Accordingly, it is now well accepted that regular physical exercise offers an effective therapeutic intervention to improve insulin action in skeletal muscle in insulin‐resistant individuals. This review provides evidence that physical inactivity is significantly associated with IGT and directly contributes to the cascade of events that lead to the expression of the ‘exercise‐deficient phenotype’ associated with insulin resistance and type 2 diabetes. In contrast, exercise training will be shown to significantly reduce the risk of developing insulin resistance by improving glucose tolerance and insulin action in individuals predisposed to develop type 2 diabetes. Several putative mechanisms for enhanced glucose uptake after exercise training will be discussed. A determination of the underlying biological mechanisms that result from exercise training is essential in order to define the precise variations in physical activity that result in the most desired effects on targeted risk factors, and to aid in the development of such interventions. Copyright © 2004 John Wiley & Sons, Ltd.
Diminished transcription of chromosome arm 4q is inversely related to local spreading of hepatocellular carcinoma
Authors: Dykema Karl J. , Furge Kyle A.
Published: August 2004

To identify transcriptional features associated with local spreading of hepatocellular carcinoma (HCC), regional expression biases were determined from the gene expression profiles of solitary HCC tumors and of HCC tumors associated with additional intrahepatic lesions and/or portal vein involvement. Regional expression biases are genetic intervals in which gene expression is coordinately changed. Often, but not always, a regional expression bias indicates an underlying chromosomal aberration. This study demonstrated that the presence or absence of a regional expression bias on chromosome arm 4q can predict the presence or absence of intrahepatic spread/portal vein involvement in 83% of cases analyzed (n = 40). These results suggest that the transcriptional state of 4q may be a marker for local spreading of HCC and that a more detailed genetic/epigenetic characterization of this locus may provide additional insights into HCC development. © 2004 Wiley‐Liss, Inc.
Determination of the Dimerization Equilibrium Constants of Omeprazole and Pirkle's Alcohol through Optical‐Rotation Measurements
Authors: Baciocchi Renato , Juza Markus , Classen Jörg , Mazzotti Marco , Morbidelli Massimo
Published: August 2004

A new procedure to measure the equilibrium constants for the dimerization (homochiral and heterochiral) reactions of enantiomers in solution was applied to two different compounds, namely omeprazole and Pirkle's alcohol, both in CHCl3. This procedure is based on the measurement of the optical rotation of the solution as a function of its composition, which exhibits a nonlinear dependence on the enantiomer enrichment. Such nonlinearity depends on the extent of dimerization, and it is a strong effect in the case of omeprazole, whose equilibrium constants are 14.0±3.5 l/mol and 25.2±4.0 l/mol for the formation of homochiral and of heterochiral dimers, respectively. Pirkle's alcohol exhibits a weaker effect that allows only to estimate the order of magnitude of these constants, i.e., ca. 0.10 and 0.34 l/mol, respectively.
Efficient Synthesis of α,β‐Epoxy Carbonyl Compounds in Acetonitrile: Darzens Condensation of Aromatic Aldehydes with Ethyl Chloroacetate
Authors: Wang Zong‐Ting , Xu Li‐Wen , Xia Chun‐Gu , Wang Han‐Qing
Published: August 2004

The Darzens reaction of ethyl 2‐chloroacetate (1) with aromatic aldehydes 2 in the presence of polymer‐supported or nonsupported quaternary ammonium salts proceeds under mild conditions by phase‐transfer catalysis to give the corresponding epoxides 3 in satisfactory yields (Tables 1 and 2). With both MeCN as solvent and polystyrene‐supported catalysts, diastereoselective Darzens reactions proceed in excellent yields and short times, with a fair degree of stereoinduction.
Three New Homologous 3‐Alkyl‐1,4‐benzoquinones from the Fruiting Bodies of Daldinia concentrica
Authors: Qin Xiang‐Dong , Liu Ji‐Kai
Published: August 2004

A new homologous series of 3‐alkyl‐5‐methoxy‐2‐methyl‐1,4‐benzoquinones (1–3), with chain lengths of C21 to C23, were isolated from the fruiting bodies of Daldinia concentrica, together with five known compounds. The molecular structures were established by spectroscopic methods.
Aurophilic towards H‐Bonding Interactions in Phosphine‐pyrazolato‐gold(I) Complexes: Luminescence Studies and Crystal Structure of {3,5‐Bis[4‐(octyloxy)phenyl]‐1H‐pyrazolato‐κN1}(triphenylphosphine)gold{3,5‐Bis[4‐(octyloxy)phenyl]‐1H‐pyrazole} ([Au(pz)(PPh3)]⋅(Hpz))
Authors: Ovejero Paloma , Cano Mercedes , Campo José A. , Heras José V. , Laguna Antonio , Crespo Olga , Pinilla Elena , Torres M. Rosario
Published: August 2004

Compounds [{Au(pz)(PPh3)}2] (pz=3,5‐disubstituted pyrazolato; R=BuOC6H4 (bp), 1; R=C8H17OC6H4 (op), 2; R=C6H13OC6H4 (hp), 3) were easily obtained by reaction of [Au(NO3)(PPh3)] and the corresponding pyrazolato ligand. They exhibit similar NMR patterns, their ESI‐MS or FAB‐MS data being in agreement with a monomeric molecular formulation [Au(pz)(PPh3)]. By contrast, 1 crystallizes as the dimer [{Au(pz)(PPh3)}2] and exhibits aurophilic Au ⋅⋅⋅Au contacts, while crystallization of 2 in CH2Cl2/hexane gives rise to compound 4, [Au(pz)(PPh3)]⋅(Hpz). The X‐ray structure of 4 was solved, showing a H‐bond interaction between the monomer of 2 and Hpz. Reactions between [{Au(pz)(PPh3)}2] and their corresponding 1H‐pyrazole (Hpz) were also explored. Compounds 1–3 exhibit luminescence in the solid state. Their emission spectra are reported and analyzed.
Der Mechanismus der Alken‐Ozonolyse – eine kritische Betrachtung
Authors: Schank Kurt
Published: August 2004

The Mechanism of Alkene Ozonolysis – a Critical Examination. Alkene ozonolyses have been carried out in experimental organic chemistry for more than a century, and a lucid description of the underlying reaction mechanism has been developped and integrated in most chemistry textbooks. Nevertheless, there are a great number of exceptions hard to rationalize by the accepted mechanism. Attempts in the past to give more‐convincing answers to this problem failed. Therefore, the search for an alternative mechanistic model without contradictions is compelling. The present paper offers a redox model based upon the known properties of ozone both as a strong oxidant and an O‐atom‐transferring agent, especially when reacted with alkenes of sufficiently high electron density to act as reducing partners.
Phosphorus‐Nitrogen Compounds: Novel Spirocyclic Phosphazene Derivatives. Structure of 3,3″‐Propane‐1,3‐diylbis[4′,4′,6′,6′‐tetrachloro‐3,4‐dihydrospiro[1,3,2‐benzoxazaphosphorine‐2,2′λ5‐[4λ5,6λ5][1,3,5,2,4,6]triazatriphosphorine]]
Authors: Bilge Selen , Natsagdorj Amgalan , Demiriz Şemsay , Çaylak Nagihan , Kılıç Zeynel , Hökelek Tuncer
Published: August 2004

The reactions of N2O2‐donor‐type aminopodands1–3with trimer N3P3Cl6led to the novel spirocyclic phosphazene derivatives4–10(Scheme). Compounds4–7and8–10are the first examples of the substituted spiro‐ansa‐spiro and spiro‐bino‐spiro phosphazene derivatives, respectively. The pyrrolidinyl‐substituted phosphazene derivatives7and10were synthesized from5and9, respectively, with an excess of pyrrolidine. The reaction of aminopodand3(R(CH2)4) with N3P3Cl6in dry THF afforded only spiro‐ansa‐spiro phosphazene6. The molecular structure of compound9was determined by X‐ray diffraction: it shows the novel spiro‐bino‐spiro phosphazene architecture.
Seven New Norditerpenoid Alkaloids from Spanish Consolida orientalis
Authors: Alva Alenguer , Grandez Maritza , Madinaveitia Alberto , de la Fuente Gabriel , Gavín José A.
Published: August 2004

Seven new norditerpenoid alkaloids named 1‐O‐demethyltricornine (1), 14‐O‐benzoyltakaosamine (2), 1‐O,19‐didehydrotakaosamine (3), 14‐O‐demethyldelboxine (4), all belonging to the lycoctonine‐type alkaloids, and 8‐O‐methylconsolarine (5), 14‐O‐deacetylpubescenine (6), and 18‐O‐benzoyl‐14‐O‐deacetyl‐18‐O‐demethylpubescenine (7), these belonging to the rare group of the 6‐epilycoctonine‐type alkaloids, were isolated from the aerial parts of Consolida orientalis (Gay) Schröd. The structures of the new compounds were elucidated by 1D‐ and 2D‐NMR and HR‐EI‐MS. Moreover, 37 known norditerpenoid alkaloids (lycoctonine‐type) were isolated and identified by comparing their spectral data with those reported in the literature.
Voltammetry as a Virtual Potentiometric Sensor in Modelling of a Metal/Ligand System and Refinement of Stability Constants. Part 2
Authors: Cukrowski Ignacy , Zhang Jian Min , van Aswegen Anton
Published: August 2004

A mathematical conversion of data from nonequilibrium and dynamic voltammetric techniques (direct‐current‐sampled (DC) and differential‐pulse (DP) polarography) into potentiometric, free‐metal‐ion sensor‐type data is described and employed in the study of BiIII complexes with the ligand picolinic acid (=pyridine‐2‐carboxylic acid) (labile and dynamic metal/ligand system). A novel procedure that allows evaluation of experimental data collected at very low pH values (acid‐base titration) is proposed. Software ESTA dedicated to potentiometry was successfully employed in the refinement operations performed with virtual potentiometric (VP) data obtained from DC and DP polarography, the latter being performed at fixed pH (ligand titration) as well as at fixed [LT]/[MT] ratio (acid‐base titration). It was possible to refine stability constants either separately from VP‐DC or VP‐DP, or simultaneously from any combination of VP‐DC and VP‐DP obtained from ligand and/or acid‐base titrations. The concept of VP‐DC or VP‐DP is employed for the first time in the study of an unknown BiIII/picolinic acid/OH system, and numerous documented and possible advantages are discussed. Five complexes of bismuth, ML, ML2, ML3, ML4, and ML3(OH) and their stability constants (as log β) 7.48±0.01, 13.94±0.01, 18.10±0.04, 20.47±0.25, and 26.65±0.03, respectively, are reported at 0.5M (Na,H)NO3 ionic strength and T 298 K. The proposed procedure can be easily utilized also by non‐electrochemists who are interested in, e.g., ligand‐design strategies.
Small‐animal models for testing macroporous ceramic bone substitutes
Authors: Le Guehennec L. , Goyenvalle E. , Aguado E , Houchmand‐Cuny M. , Enkel B. , Pilet P. , Daculsi G. , Layrolle P.
Published: August 2004

The aim of this study was to compare the bone colonization of a macroporous biphasic calcium phosphate (MBCP) ceramic in different sites (femur, tibia, and calvaria) in two animal species (rats and rabbits). A critical size defect model was used in all cases with implantation for 21 days. Bone colonization in the empty and MBCP‐filled defects was measured with the use of backscattered electron microscopy (BSEM). In the empty cavities, bone healing remained on the edges, and did not bridge the critical size defects. Bone growth was observed in all the implantation sites in rats (approx. 13.6–36.6% of the total defect area, with ceramic ranging from 46.1 to 51.9%). The bone colonization appeared statistically higher in the femur of rabbits (48.5%) than in the tibia (12.6%) and calvaria (22.9%) sites. This slightly higher degree of bone healing was related to differences in the bone architecture of the implantation sites. Concerning the comparison between animal species, bone colonization appeared greater in rabbits than in rats for the femoral site (48.5% vs. 29.6%). For the other two sites (the tibia and calvaria), there was no statistically significant difference. The increased bone ingrowth observed in rabbit femurs might be due to the large bone surface area in contact with the MBCP ceramics. The femoral epiphysis of rabbits is therefore a favorable model for testing the bone‐bonding capacity of materials, but a comparison with other implantation sites is subject to bias. This study shows that well‐conducted and fully validated models with the use of small animals are essential in the development of new bone substitutes. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 72B: 69–78, 2005
Analytical technique for quantification of selected resorbable calcium phosphate bone void fillers with the use of polarized‐light microscopy
Authors: Clineff Theodore D. , Erbe Erik M. , Bauer Thomas W. , Carroll Beth E.
Published: August 2004

Synthetic calcium phosphate bone void fillers promote varying rates of bone formation and material resorption depending on chemistry, porosity, pore structure, and implant site. The objective of this study was to quantify the resorption of a novel ultraporous β‐tricalcium phosphate cancellous bone void filler with simultaneous quantification of bone formation in a canine humerus model. Potential measurement error involved in conventional histomorphometry using Von Kossa stains inspired the development of a new technique. This technique utilizes bright‐field and polarized‐light microscopy in conjunction with image analysis software, allowing more accurate histomorphometry. This technique was validated with two separate controlled experiments. Scanning electron microscopy further supported the results. The findings suggest that the use of polarized‐light microscopy combined with image analysis software can be an effective tool in simultaneously quantifying calcium phosphate resorption and bone formation. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 72B: 125–130, 2005
Quantum‐connectivity descriptors in modeling solubility of environmentally important organic compounds
Authors: Estrada Ernesto , Delgado Eduardo J. , Alderete Joel B. , Jaña Gonzalo A.
Published: August 2004

Quantum‐connectivity indices are topographic descriptors combining quantum‐chemical and topological information. They are used to describe the water solubility of a noncongeneric data set of organic compounds. A QSPR model is obtained with two quantum‐connectivity indices that accounts for more than 90% of the variance in the water solubility of these chemicals. This model is compared to other five QSPR models using constitutional, electrostatic, geometric, quantum‐chemical, and topological descriptors calculated by CODESSA. None of these models accounts for more than 85% of the variance in water solubility of the compounds in this data set. The QSPR model obtained with quantum‐connectivity indices is also better than that generated from the general pool of 508 CODESSA indices. Models with up to five variables were explored and compared with the model obtained here. It is shown that quantum‐connectivity indices contain more structural information than other classes of descriptors at least for describing the water solubility of these 53 chemicals. Structural interpretation of the QSPR model developed as well as the role of the quantum‐connectivity indices included in it are also analyzed. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 1787–1796, 2004
Jason Wagoner and Nathan A. Baker, "Solvation forces on biomolecular structures: A comparison of explicit solvent and Poisson‐Boltzmann models,"Journal of Computational Chemistry(2004) 25(13) 1623–1629
Authors: Wagoner Jason , Baker Nathan A.
Published: August 2004

The article to which this Erratum refers was published in Journal of Computational Chemistry (2004) 25(13) 1623–1629
Altered regulation of EGF receptor signaling following a partial hepatectomy
Authors: Skarpen E. , Oksvold M.P. , Grøsvik H. , Widnes C. , Huitfeldt H.S.
Published: August 2004

We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre‐replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC‐regulated T654‐site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654‐phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF‐mediated EGFR autophosphorylation, altered ligand‐induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR‐Ras‐Raf‐ERK pathway. Further, we found that both TPA and a PH enhanced EGF‐induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase‐activity. This may be a pre‐replicative competence event that increases growth factor effects during G1 progression. © 2004 Wiley‐Liss, Inc.
Interleukin (IL)‐1β toxicity to islet β cells: Efaroxan exerts a complete protection
Authors: Papaccio Gianpaolo , Graziano Antonio , Valiante Salvatore , D'Aquino Riccardo , Travali Salvatore , Nicoletti Ferdinando
Published: August 2004

Interleukin (IL)‐1β‐treated rat islets of Langerhans were exposed in vitro either to the imidazoline compound, Efaroxan, or to the selective inducible nitric oxide synthase (iNOS) inhibitor, 1400W, in a medium containing a high concentration of glucose (16.7 mmol/L). Our data have evidenced the following: (i) addition of Efaroxan to islet cultures inhibited IL‐1β activation of ICE (cysteine protease IL‐1β converting enzyme) while addition of 1400W did not; (ii) Efaroxan completely inhibited IL‐1β‐induced suppression of insulin secretion and induction of iNOS mRNA transcripts, and, in addition, counteracted islet β‐cell protein profile alterations, Bax‐cytochrome c translocation, caspase activation, and apoptosis; (iii) 1400W inhibited IL‐1β induction of iNOS, but failed to completely counteract the other cytotoxic effects; (iv) the two compounds, moreover, exerted different effects on manganese superoxide dismutase (MnSOD), in fact, while Efaroxan inhibited the early stimulatory effect of IL‐1β on MnSOD, 1400W did not. Thus, Efaroxan completely protected islet β cells from damage caused by IL‐1β‐induced toxicity, while compound 1400W only inhibited NO radical production without altering the cytokine's cytotoxicity. Our observations have evidenced that suppression of ICE activation is required to counteract IL‐1β‐mediated islet β cell toxicity, and that IL‐1β‐induced apoptosis is NO‐independent and involves the cytochrome c‐mitochondrial pathway. © 2004 Wiley‐Liss, Inc.
Fermentation and oxygen transfer characteristics in serine alkaline protease production by recombinant Bacillus subtilis in molasses‐based complex medium
Authors: Çalık Güzide , Pehlivan Nur , Özçelik İlknur Şenver , Çalık Pınar , Özdamar Tunçer H
Published: August 2004

Serine alkaline protease (SAP) production in a complex medium based on physically pretreated molasses by recombinant Bacillus subtilis carrying pHV1431::subC gene is described. The effects of oxygen transfer were investigated in 3.5 dm3 bioreactor systems with controls for agitation rate, dissolved oxygen, pH, temperature, and foam formation under two different agitation rates, ie N = 500 and 750 min−1, and four different air flow rates, ie Q/VR = 0.2, 0.5, 0.7, and 1.0 vvm, at a molasses concentration equivalent to initial sucrose concentration (CSo) of 20 kg m−3. The yield values (YX/S, YX/O, YS/O) and maintenance coefficient of oxygen (mO), were calculated. mO decreased with the increase in the air‐inlet rate. Increase in oxygen transfer rate increased the rate of growth and SAP activity, and affected the cultivation time to achieve maximum expression of SAP activity. At Q/VR = 0.5 vvm and N = 750 min−1, SAP activity reached 2250 U cm−3 at t = 36 h. The oxygen transfer coefficient (KLa) and oxygen uptake rate (−rO) were measured throughout the fermentations and their variation with the oxygen transfer conditions determined. New correlations for the calculation of KLa and −rO are proposed. Copyright © 2004 Society of Chemical Industry
Recovery and reuse of chromium from tannery wastewaters using Turbinaria ornata seaweed
Authors: Aravindhan Rathinam , Madhan Balaraman , Rao Jonnalagadda Raghava , Nair Balachandran Unni
Published: August 2004

Brown seaweed (Turbinaria spp) was pre‐treated with sulfuric acid, calcium chloride and magnesium chloride and tested for its ability to remove chromium from tannery wastewater. Protonated seaweeds gave better uptake of chromium compared with calcium and magnesium treatments. Chromium uptake was optimal at pH 3.5. Turbinaria weed exhibited maximum uptake of about 31 mg of chromium for one gram of seaweed at an initial concentration of 1000 ppm of chromium. Freundlich and Langmuir adsorption isotherm models were used to describe the biosorption of chromium(III) by Turbinaria spp. The chromium‐loaded seaweed was reused as a reductant in the preparation of the tanning agent basic chromium sulfate (BCS). Leathers made from this tanning agent had properties comparable to conventionally processed chrome‐tanned leathers. Copyright © 2004 Society of Chemical Industry
Changes of gene expression profiles during neuronal differentiation of central nervous system precursors treated with ascorbic acid
Authors: Yu Dong‐Hyun , Lee Ki‐Hwan , Lee Ji‐Yeon , Kim Sujong , Shin Dong‐Mi , Kim Jin‐Hyuk , Lee Young‐Seek , Lee Yeon‐Sook , Oh Sun Kyung , Moon Shin Yong , Lee Sang‐Hun , Lee Yong‐Sung
Published: August 2004

Ascorbic acid (AA) has been shown to increase the yield of dopaminergic (DA) neurons derived from basic fibroblast growth factor (bFGF)‐expanded mesencephalic precursors. To understand the molecular mechanisms underlying this phenomenon, we used cDNA microarray analysis to examine differential expression of neuronal genes following AA treatment. The putative precursor cells were isolated from E13 rat ventral mesencephalons and expanded in the presence of bFGF. Cells were incubated in mitogen‐free media supplemented with 200 μM AA or were left untreated as a control, and total RNA was isolated at different time points (expansion stage and 1, 3, and 6 days after induction of differentiation) and subjected to cDNA microarray analysis. Differentiation was evaluated by Western blot analysis and immunocytochemistry of neuron‐specific markers. AA treatment of the mesencephalic precursors increased the expression of neuronal (MAP2) and astrocytic (glial fibrillary acidic protein) markers and the percentage of tyrosine hydroxylase (TH)‐positive cells. The microarray analysis revealed that 12 known genes were up‐regulated and 20 known genes were down‐regulated in expansion‐stage AA‐treated cells. Six days after the induction of differentiation, AA‐treated cells showed up‐regulation of 48 known genes and down‐regulation of 5 known genes. Our results identified several proteins, such as transferrin, S‐100, and somatostatin, as being differentially regulated in AA‐treated mesencephalic precursors. This novel result may lead to a better understanding of the molecular mechanisms underlying the AA‐induced differentiation of mesencephalic precursors into DA neurons and may form the basis for improved DA neuronal production for treatment of Parkinson's disease patients. © 2004 Wiley‐Liss, Inc.
Mitochondria‐derived oxidative stress induces a heat shock protein response
Authors: Barrett Matthew J. , Alones Vinessa , Wang Kai Xuan , Phan Lien , Swerdlow Russell H.
Published: August 2004

In addition to minimizing native and non‐native protein aggregations, heat shock proteins (HSPs) regulate programmed cell death pathways. Members of this conserved protein family are in general activated by oxidative stress, and likely help maintain viability under this stress condition. To further our understanding of heat shock protein physiology, we studied whether a specific subtype of oxidative stress, namely that arising from mitochondrial electron transport chain inhibition, induced a heat shock protein response. We exposed human teratocarcinoma (NT2) cells to varying concentrations of the cytochrome oxidase inhibitor sodium azide. Micromolar exposures resulted in a cytoplasm to nucleus translocation of the inducible Hsp70 and of Hsp40, and this was followed by an overall upregulation. The response did not coincide temporally with the onset of azide exposure, but rather was activated when the degree of cytochrome oxidase inhibition (which was progressive over time) surpassed a threshold. Azide did not affect either Hsp70 or Hsp40 dynamics in NT2 ρ0 cells, which lack functional electron transport chains. For the azide‐exposed native cell line, addition of the antioxidant trolox to the medium abrogated both Hsp70/Hsp40 translocation and upregulation. We conclude that mitochondrial electron transport chain dysfunction activates a heat shock protein response, and that this response is mediated by oxidative stress. © 2004 Wiley‐Liss, Inc.
N‐acetylcysteine prevents endotoxin‐induced degeneration of oligodendrocyte progenitors and hypomyelination in developing rat brain
Authors: Paintlia Manjeet K. , Paintlia Ajaib S. , Barbosa Ernest , Singh Inderjit , Singh Avtar K.
Published: August 2004

Periventricular leukomalacia (PVL), the dominant form of brain injury in premature infants, is characterized by diffuse white matter injury and is associated with cerebral palsy (CP). Maternal and placental infections are major causes of prematurity and identifiable etiology of PVL and CP. Here we have evaluated the therapeutic efficacy of N‐acetylcysteine (NAC), a potent antioxidant and precursor of glutathione, to attenuate lipopolysaccharide (LPS)‐induced white matter injury and hypomyelination in the developing rat brain, an animal model of PVL. Intraperitoneal pretreatment of pregnant female rats with NAC (50 mg/kg), 2 hr prior to administration of LPS at embryonic day 18 (E18), attenuated the LPS‐induced expression of inflammatory cytokines such as tumor necrosis factor‐α, interleukin‐1β, and inducible nitric oxide synthase in fetal rat brains. There were significantly reduced numbers of TUNEL+ nuclei coimmunostained for platelet‐derived growth factor‐αR+ [a surface marker for oligodendrocyte progenitor cells (OPCs)] at E20 in the subventricular zone of fetal rat brain in the NAC + LPS group compared with the untreated LPS group. Interestingly, immunostaining for O4 and O1 as markers for late OPCs and immature oligodendrocytes demonstrated fewer O4+ and O1+ cells in the LPS group compared with the NAC + LPS and control groups. Consistent with O4+/O1+ cell counts, the expression of myelin proteins such as myelin basic protein, proteolipid protein, and 2′3′‐cyclic nucleotide phosphodiesterase, including transcription factors such as MyT1 and Gtx, was less in the LPS group at late postnatal days, indicating severe hypomyelination in the developing rat brain when compared with NAC + LPS and control groups. Collectively, these data support the hypothesis that NAC may provide neuroprotection and attenuate the degeneration of OPCs against LPS evoked inflammatory response and white matter injury in developing rat brain. Moreover, these data suggest the possible use of NAC as a treatment for pregnant women with maternal or placental infection as a means of minimizing the risk of PVL and CP. © 2004 Wiley‐Liss, Inc.
Ternary complex with Trk, p75, and an ankyrin‐rich membrane spanning protein
Authors: Chang Mi‐Sook , Arevalo Juan Carlos , Chao Moses V.
Published: August 2004

Neurotrophins play many critical roles in regulating neuronal plasticity, survival, and differentiation in the nervous system. Neurotrophins recognize two different receptors, the Trk receptor tyrosine kinase and the p75 neurotrophin receptor, which are associated closely. Several adaptor proteins are associated with each receptor. An ankyrin‐rich membrane spanning protein (ARMS), originally identified as a substrate for protein kinase D (Kidins220) and as a p75 interacting protein, serves as a novel downstream target of Trk receptor tyrosine kinases. Kidins220/ARMS is co‐expressed frequently with Trk and p75 and represents the only membrane‐associated protein known to interact with both receptors. We report here that a ternary complex can be formed between Trk, p75, and Kidins220/ARMS. The extracellular domains of the TrkA and the p75 receptors are necessary for their association, whereas the juxtamembrane region of p75 was responsible for the interaction with Kidins220/ARMS. Interestingly, increasing the level of Kidins220/ARMS expression resulted in a decreased association of TrkA with p75. These findings thus suggest that Kidins220/ARMS plays an important role in regulating interactions between Trk and p75 neurotrophin receptors. © 2004 Wiley‐Liss, Inc.
Effect of activation time on the nuclear remodeling and in vitro development of nuclear transfer embryos derived from bovine somatic cells
Authors: Choi Jong‐Yeob , Kim Choung‐Ik , Park Choon‐Keun , Yang Boo‐Keun , Cheong Hee‐Tae
Published: August 2004

This study was conducted to investigate the effect of recipient activation time on the chromatin structure and development of bovine nuclear transfer embryos. Serum‐starved skin cells were electrofused to enucleated oocytes, activated 1–5 hr after fusion, and cultured in vitro. Some fused eggs were fixed at each time point after fusion without activation, or 3 or 7 hr after activation. Some nocodazole treated zygotes were fixed to analyze their chromosome constitutions. The proportion of eggs with a morphologically normal premature chromosome condensation (PCC) state increased 1–2 hr after fusion. Whereas eggs with elongated chromosome plate increased as activation time was prolonged to 3 hr, and 5 hr after fusion, 58.1% of eggs showed more than two scattered chromosome sets. The proportion of eggs with a single chromatin mass (40.6–56.7%) significantly increased when eggs were activated within 2.5 hr after fusion (P < 0.05). Only 23.3% of reconstituted embryos activated 5 hr after fusion formed one pronucleus‐like structure (PN), whereas, 64.5–78.3% of embryos activated 1–2.5 hr after fusion formed one PN. The proportion of embryos with normal chromosome constitutions decreased as activation time was prolonged. Development rates to the blastocyst stage were higher in eggs activated within 2 hr after fusion (17.3–21.7%) compared to those of others (0–8.6%, P < 0.05). The result of the present study suggests that activation time can affect the chromatin structure and in vitro development of bovine nuclear transfer embryos. Mol. Reprod. Dev. 69: 289–295, 2004. © 2004 Wiley‐Liss, Inc.
Expression of peroxiredoxins in bovine oocytes and embryos produced in vitro
Authors: Leyens Gregory , Knoops Bernard , Donnay Isabelle
Published: August 2004

Peroxiredoxins (PRDXs) form a family of peroxidases involved in antioxidant protection and cell signaling. Due to their peroxide reductase activity, these enzymes might be involved in fine‐tuning peroxide levels in embryos during in vitro production. In this study, RT‐PCR was used to examine the expression of the six PRDX isoforms (PRDX1 to PRDX6) in bovine oocytes and embryos. PRDXs were detected in oocytes both before and after in vitro maturation. Besides, PRDX6 was up‐regulated after maturation. Single embryos were analyzed from the two‐cell to the blastocyst stages. PRDX1 and PRDX5 transcripts were detected throughout development. PRDX2, PRDX3, and PRDX6 were not expressed around the 9‐ to 16‐cell stage. PRDX4 transcripts were weakly detected in pools of embryos from the 9‐ to 16‐cell stage onwards. In situ immunodetection of PRDX5, which was previously reported to exhibit the widest subcellular distribution among PRDXs in adult mammalian cells, showed a mitochondrial distribution pattern in the bovine embryo. Finally, the potential modulation by oxidative stress of PRDX expression around the major embryonic genome activation was evaluated by culturing embryos under 20% O2 instead of 5%. No significant difference in the pattern of PRDX expression was observed under 20% O2. In conclusion, our data show for the first time that PRDXs are expressed in mammalian oocytes and early embryos. Moreover, the bovine transcripts exhibit various patterns of expression that might be related to the potential role of PRDXs in oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 243–251, 2004. © 2004 Wiley‐Liss, Inc.
The generalized global basis (GGB) method
Authors: Waisman H. , Fish J. , Tuminaro R. S. , Shadid J.
Published: August 2004

In this work, we present the generalized global basis (GGB) method aimed at enhancing performance of multilevel solvers for difficult systems such as those arising from indefinite and non‐symmetric matrices. The GGB method is based on the global basis (GB) method (Int J Numer Methods Eng 2000; 49:439–460, 461–478), which constructs an auxiliary coarse model from the largest eigenvalues of the iteration matrix. The GGB method projects these modes which would cause slow convergence to a coarse problem which is then used to eliminate these modes. Numerical examples show that best performance is obtained when GGB is accelerated by GMRES and used for problems with multiple right‐hand sides. In addition, it is demonstrated that GGB method can enhance restarted GMRES strategies by retention of subspace information. Copyright © 2004 John Wiley & Sons, Ltd.
Anti‐CD52 antibody, alemtuzumab, binds to Langerhans cells in Langerhans cell histiocytosis
Authors: Jordan Michael B. , McClain Kenneth L. , Yan Xiaotian , Hicks John , Jaffe Ronald
Published: August 2004

Background

The humanized anti‐CD52 monoclonal antibody, alemtuzumab (or Campath‐1H), has been shown to potently deplete lymphocytes in human patients. It has been used to successfully treat graft versus host disease and chronic lymphocytic leukemia (CLL). CD52 is expressed on normal lymphocytes, monocytes, and some dendritic cell subsets. However, normal Langerhans cells (LC's) in the skin do not bind alemtuzumab. We sought to determine whether the pathologic LC's of Langerhans cell histiocytosis (LCH) express CD52 and thus could be targeted by this antibody.

Methods

Immunohistochemistry was performed on both frozen and fixed/paraffin‐embedded tissue specimens using either Campath‐1G (the parental rat isotype) or Campath‐1H (the humanized version of Campath in clinical use).

Results

Both Campath‐1H and Campath‐1G were found to bind to the pathologic LC's in LCH, but not the normal LC's of skin. Specific staining was demonstrated in all (13 of 13) LCH specimens examined, though staining was somewhat variable among specimens, and tended to be weaker in paraffin‐embedded specimens.

Conclusions

Expression of CD52 by the pathologic LC's seen in LCH suggests that alemtuzumab may represent a new, targeted therapy for this disease. Such therapy is particularly needed for patients with refractory, high‐risk disease. Further investigation of the possible clinical use of this antibody in these patients is warranted. © 2004 Wiley‐Liss, Inc.
Proteomic analysis of mitochondrial proteins in cardiomyocytes from chronic stressed rat
Authors: Liu Xiao‐Hua , Qian Ling‐Jia , Gong Jing‐Bo , Shen Jing , Zhang Xue‐Min , Qian Xiao‐Hong
Published: August 2004

Chronic restraint stress induces cardiac dysfunction as well as cardiomyocyte injury including severe ultrastructural alteration and cell death, but its mechanism and molecular basis remain unclear. Mitochondria play a key role in regulating cell life. For exploring mitochondrial proteins which correlate with stress‐induced injury, two‐dimensional electrophoresis and matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF MS) were applied. After comparing the protein profiles of myocardial mitochondria between a chronic restraint stress group and a control group, 11 protein spots were found altered, seven of which were identified by MALDI‐TOF MS. Among the seven proteins, five proteins involved in the Krebs cycle and lipid metabolism in mitochondria decreased after chronic restraint stress. They were identified as carnitine palmitoyltransferase 2, mitochondrial acyl‐CoA thioesterase 1, isocitrate dehydrogenase 3 (NAD+) alpha, fumarate hydratase 1 and pyruvate dehydrogenase beta. The last two proteins, creatine kinase and prohibitin, increased after chronic restraint stress. Biochemical tests for energy metabolism in mitochondria also supported the proteomic results. These findings provide clues for understanding the mechanism of dysfunction or injury in cardiomyocytes induced by chronic stress.
Living polymerization of styrene initiated by mercaptan/ε‐caprolactam
Authors: Hu Yu‐Hsiang , Chen Chuh‐Yung , Wang Chen‐Chien , Huang Yao‐Hui , Wang Shao‐Ping
Published: August 2004

The bulk polymerization of styrene initiated by ϵ‐caprolactam (CL) and n‐dodecyl mercaptan (RSH) has been explored. This novel polymerization system shows living characteristics. For example, the molecular weight of the resulting polymers increases with conversion, and the system has the ability to form diblock copolymers and so forth. The polymer chain end contains thiol and lactam structures, which we have investigated with Fourier transform infrared, 1H NMR, and 13C NMR techniques. Electron spin resonance spectra and theoretical calculations by the Hartree–Fock methods have been used to examine the mechanism. The results reveal that the initial polymerization starts from thiol via a chain‐transfer reaction, and the propagation proceeds by the insertion of a monomer between the terminal group and the intermediate structure of lactam. Finally, the polymerization kinetics have been examined. The polymerization rate varies linearly with the concentration of CL and RSH, and this confirms the mechanism. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4976–4993, 2004
Novice and expert teachers' conceptions of learners' prior knowledge
Authors: Meyer Helen
Published: August 2004

This study presents comparative case studies of preservice and first‐year teachers' and expert teachers' conceptions of the concept of prior knowledge. Kelly's (The Psychology of Personal Construct, New York: W.W. Norton, 1955) theory of personal constructs as discussed by Akerson, Flick, and Lederman (Journal of Research in Science Teaching, 2000, 37, 363–385) in relationship to prior knowledge underpins the study. Six teachers were selected to participate in the case studies based upon their level experience teaching science and their willingness to take part. The comparative case studies of the novice and expert teachers provide insights into (a) how novice and expert teachers understand the concept of prior knowledge and (b) how they use this knowledge to make instructional decisions. Data collection consisted of interviews, classroom observations, and document analysis. Findings suggest that novice teachers hold insufficient conceptions of prior knowledge and its role in instruction to effectively implement constructivist teaching practices. While expert teachers hold a complex conception of prior knowledge and make use of their students' prior knowledge in significant ways during instruction. A second finding was an apparent mismatch between the novice teachers' beliefs about their urban students' life experiences and prior knowledge and the wealth of knowledge the expert teachers found to draw upon. © 2004 Wiley Periodicals, Inc. Sci Ed 88:970–983, 2004