bluuebunny/crossref_metadata_2025 huggingface dataset:
Stochastic search for isomers on a quantum mechanical surface
Authors: Saunders Martin
Published: February 2004

In studying molecules with unusual bonding and structures, it is desirable to be able to find all the isomers that are minima on the energy surface. A stochastic search procedure is described for seeking all the isomers on a surface defined by quantum mechanical calculations involving random kicks followed by optimization. It has been applied to searching for singlet structures for C6 using the restricted Hartree–Fock/6‐311G basis set. In addition to the linear chain and ring previously investigated, 11 additional structures (A–K) were located at this level. These provide a basis for discussing qualitative bonding motifs for this carbon cluster. The application of a similar idea to searching for transition states is discussed. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 621–626, 2004
A multilayered approach to approximating solute polarization
Authors: Maurer Richard I. , Reynolds Christopher A.
Published: February 2004

A hybrid multilayered “ONIOM”‐type approach to solvation is presented in which the basic free energy of hydration is taken from the Poisson Boltzmann method and the contribution to the solute polarization is taken from a quantum mechanical implementation of the Born method. The method has been tested on the 52 neutral molecules used in the AM1–SM2 parameterization, and the polarized continuum method is taken as the standard by which the results are assessed. Regression analysis shows that the method gives a small improvement over the standard Poisson Boltzmann method or a dramatic improvement over the Born method. The system presented here represents one of the more straightforward applications of the multilayered approach to solvation, but other more sophisticated approaches are discussed. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 627–631, 2004
SVMtm: Support vector machines to predict transmembrane segments
Authors: Yuan Zheng , Mattick John S. , Teasdale Rohan D.
Published: February 2004

A new method has been developed for prediction of transmembrane helices using support vector machines. Different coding schemes of protein sequences were explored, and their performances were assessed by crossvalidation tests. The best performance method can predict the transmembrane helices with sensitivity of 93.4% and precision of 92.0%. For each predicted transmembrane segment, a score is given to show the strength of transmembrane signal and the prediction reliability. In particular, this method can distinguish transmembrane proteins from soluble proteins with an accuracy of ∼99%. This method can be used to complement current transmembrane helix prediction methods and can be used for consensus analysis of entire proteomes. The predictor is located at http://genet.imb.uq.edu.au/predictors/SVMtm. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 632–636, 2004
Systematic comparison of force fields for microscopic simulations of NaCl in aqueous solutions: Diffusion, free energy of hydration, and structural properties
Authors: Patra Michael , Karttunen Mikko
Published: February 2004

In this article we compare different force fields that are widely used (Gromacs, Charmm‐22/x‐Plor, Charmm‐27, Amber‐1999, OPLS‐AA) in biophysical simulations containing aqueous NaCl. We show that the uncertainties of the microscopic parameters of, in particular, sodium, and, to a lesser extent, chloride, translate into large differences in the computed radial‐distribution functions. This uncertainty reflect the incomplete experimental knowledge of the structural properties of ionic aqueous solutions at finite molarity. We discuss possible implications on the computation of potential of mean force and effective potentials. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 678–689, 2004
A generating equation for mixing rules and two new mixing rules for interatomic potential energy parameters
Authors: Al‐Matar Ali Khalaf , Rockstraw David A.
Published: February 2004

A generating equation for the mixing rules of interatomic potential energy parameters is proposed. It is demonstrated that this equation can, indeed, reproduce many popular mixing rules. A weighting matrix is used with the generating equation. This weighting matrix approach is superior to the present status of mixing rule development. A systematic framework is given for devising new mixing rules and/or comparing them. Two new mixing rules, which are more accurate than the available rules in the literature, are proposed. These rules are capable of reproducing the collision diameter and well‐depth parameters for the binary values of noble gases to within their experimental uncertainties. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 660–668, 2004
Study of proton adsorption at heterogeneous oxide/electrolyte interface. Prediction of the surface potential using Monte Carlo simulations and 1‐pK approach
Authors: Zarzycki P. , Charmas R. , Szabelski P.
Published: February 2004

Adsorption of protons on a heterogeneous solid surface is modeled using the Monte Carlo (MC) simulation method. The surface of an oxide is assumed to consist of adsorption sites with pK assigned according to a quasi‐Gaussian distribution. The influence of the electrostatic interactions combined with the energetic heterogeneity of the surface is examined and the MC results are compared with the predictions of the analytical 1‐pK approach. The surface potential behavior is examined using both “experimental” MC results and “theoretical” results obtained from the application of 1‐pK model. The results are compared qualitatively with experimental determination of the surface potential of metal oxide surfaces. They confirm that the relation between the surface potential and the pH of bulk solution should not be described by the Nernst equation but by the equation with the parameter linearly reducing Nerstian potential. The values of this parameter are examined with respect to degree of surface energetic heterogeneity and site density of the surface. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 704–711, 2004
Importance of solvent accessibility and contact surfaces in modeling side‐chain conformations in proteins
Authors: Eyal Eran , Najmanovich Rafael , Mcconkey Brendan J. , Edelman Marvin , Sobolev Vladimir
Published: February 2004

Contact surface area and chemical properties of atoms are used to concurrently predict conformations of multiple amino acid side chains on a fixed protein backbone. The combination of surface complementarity and solvent‐accessible surface accounts for van der Waals forces and solvation free energy. The scoring function is particularly suitable for modeling partially buried side chains. Both iterative and stochastic searching approaches are used. Our programs (Sccomp‐I and Sccomp‐S), with relatively fast execution times, correctly predict χ1 angles for 92–93% of buried residues and 82–84% for all residues, with an RMSD of ∼1.7 Å for side chain heavy atoms. We find that the differential between the atomic solvation parameters and the contact surface parameters (including those between noncomplementary atoms) is positive; i.e., most protein atoms prefer surface contact with other protein atoms rather than with the solvent. This might correspond to the driving force for maximizing packing of the protein. The influence of the crystal packing, completeness of rotamer library and precise positioning of Cβ atoms on the accuracy of side‐chain prediction are examined. The Sccomp‐S and Sccomp‐I programs can be accessed through the Web (http://sgedg.weizmann.ac.il/sccomp.html) and are available for several platforms. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 712–724, 2004
Numerical examination of performance of some exchange‐correlation functionals for molecules containing heavy elements
Authors: Wang Fan , Li Lemin
Published: February 2004

The performance of 17 exchange‐correlation functionals for molecules containing heavy elements are numerically examined through four‐component relativistic density DFT calculations. The examined functionals show the similar accuracy as they do for the molecules containing light elements only except for bond lengths. LDA and OP86 produce good results for bond lengths and frequencies but bad bond energies. Different functionals do not show much different performance for bond energies except LDA. BP86 and GP86 produce results with average accuracy while LYP does not perform well. Although encouraging results are obtained with functional B97GGA‐1, other heavily parameterized and meta‐GGA functionals do not produce impressive results. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 669–677, 2004
Solvent effects on glycine II. Water‐assisted tautomerization
Authors: Balta Bülent , Aviyente Viktorya
Published: February 2004

The water‐assisted tautomerization of glycine has been investigated at the B3LYP/6‐31+G** level using supermolecules containing up to six water molecules as well as considering a 1:1 glycine–water complex embedded in a continuum. The conformations of the tautomers in this mechanism do not display an intramolecular H bond, instead the functional groups are bridged by a water molecule. The replacement of the intramolecular H bond by the bridging water reduces the polarity of the NH bond in the zwitterion and increases that of the OH bond in the neutral, stabilizing the zwitterion. Both the charge transfer effects and electrostatic interactions stabilize the nonintramolecularly H‐bonded zwitterion conformer over the intramolecularly hydrogen bonded one. The nonintramolecularly H‐bonded neutral is favored only by charge transfer effects. Although there is no strong evidence whether the intramolecularly hydrogen bonded or non hydrogen bonded structures are favored in the bulk solution represented as a dielectric continuum, it is likely that the latter species are more stable. The free energy of activation of the water‐assisted mechanism is higher than the intramolecular proton transfer channel. However, when the presumably higher conformational energy of the zwitterion reacting in the intramolecular mechanism is taken into account, both mechanisms are observed to compete. The various conformers of the neutral glycine may form via multiple proton transfer reactions through several water molecules instead of a conformational rearrangement. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 690–703, 2004
Simple algorithm for isothermal–isobaric molecular dynamics
Authors: Stern Harry A.
Published: February 2004

We review principles of non‐Hamiltonian statistical mechanics and present a new set of equations and integration algorithm for isothermal–isobaric dynamics. The chief advantage of the present scheme is that it is somewhat simpler than previous methods. We perform numerical simulations to test the accuracy of the algorithm and compare its stability to that of a “gold standard,” a symplectic integrator for Hamiltonian dynamics of the same system. The stability of the isothermal–isobaric algorithm is comparable to the stability of the symplectic integrator. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 749–761, 2004
Accurate and fast algorithm of the molecular incomplete gamma function with a complex argument
Authors: Ishida Kazuhiro
Published: February 2004

Several efficient algorithms for the accurate and fast calculation of the molecular incomplete gamma function Fm(z) with a complex argument z are developed. The complex incomplete gamma function is arising in molecular integrals over the gauge‐including atomic orbitals. Two kinds of algorithms are recommended: (1) a high‐precision version and (2) a fast version. The high‐precision version is able to guarantee 15 significant figures (10−15 in the relative error) and the fast version is able to guarantee 12 significant figures (10−12 in the relative error), at worst, within the double‐precision arithmetic. The fast version is about 5–20 times faster than the high‐precision version. For most molecular calculations, the fast version will give a satisfied precision. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 739–748, 2004
Nature of bonding in the sulfuryl group
Authors: Chesnut D. B. , Quin L. D.
Published: February 2004

The SO sulfuryl bond in a number of representative sulfoxides and sulfones has been studied at the B3LYP/6‐311+G(d,p) level in the atoms‐in‐molecules (AIM) approach involving the AIM delocalization index and the Cioslowski–Mixon localized orbitals and associated covalent bond order. The sulfuroxygen covalent bond is strongly polarized toward oxygen and the oxygen lone pairs provide significant backbonding to create short and strong SO bonds, similar in nature to those found in the analogous phosphoryl (PO) bond. Although the sulfoxides in general have larger delocalization indices than the sulfones, there is no correlation between these quantities and the bond dissociation energies. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 734–738, 2004
Intramolecular CH…Ccarbene hydrogen bonds and competing interactions in monoprotonated tripodal carbenes
Authors: Howard Siân T. , Abernethy Colin D.
Published: February 2004

The anionic tripodal N‐heterocyclic carbene (C3N2H3)3BH− first prepared by Fehlhammer, together with three neutral variants, (C3N2H3)3CH, (C3N2H3)3P, and (C3N2H3)3SiH, have been studied using quantum chemical methods. Isodesmic reactions are used to deduce that the phosphine‐bridgehead species in particular has a large‐resonance stabilization energy. All the podands undergo substantial conformational change on excitation to the lowest triplet electronic state, with effective localization of the excitation on one of the heterocyclic rings, dearomatizing it. On monoprotonation of the ground states, three of these species display intramolecular C‐H…Ccarbene hydrogen bonding: The nature and strength of these interactions is explored using model (intermolecularly hydrogen‐bonded) complexes, isodesmic reactions, and GIAO calculations of chemical shifts. One surprising result is that C‐H…Ccarbene hydrogen bonds involving ethenic hydrogens can be almost as strong as those involving the imidazolium proton (first identified by Arduengo). The case of the monoprotonated carbon bridgehead species is in particular intriguing. It is stabilized by a competitive Ccarbene…N interaction of sufficient strength to override the C‐H…Ccarbene bonding motif observed in the other structures. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 649–659, 2004
TDDFT investigation on nucleic acid bases: Comparison with experiments and standard approach
Authors: Shukla M.K. , Leszczynski Jerzy
Published: February 2004

A comprehensive theoretical study of electronic transitions of canonical nucleic acid bases, namely guanine, adenine, cytosine, uracil, and thymine, was performed. Ground state geometries were optimized at the MP2/6‐311G(d,p) level. The nature of respective potential energy surfaces was determined using the harmonic vibrational frequency analysis. The MP2 optimized geometries were used to compute electronic vertical singlet transition energies at the time‐dependent density functional theory (TDDFT) level using the B3LYP functional. The 6‐311++G(d,p), 6‐311(2+,2+)G(d,p), 6‐311(3+,3+)G(df,pd), and 6‐311(5+,5+)G(df,pd) basis sets were used for the transition energy calculations. Computed transition energies were found in good agreement with the corresponding experimental data. However, in higher transitions, the Rydberg contaminations were also obtained. The existence of πσ* type Rydberg transition was found near the lowest singlet ππ* state of all bases, which may be responsible for the ultrafast deactivation process in nucleic acid bases. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 768–778, 2004
Changes in macrophage function and morphology due to biomedical polyurethane surfaces undergoing biodegradation
Authors: Matheson Loren A. , Santerre J. Paul , Labow Rosalind S.
Published: February 2004

Monocytes are recruited to the material surface of an implanted biomedical device recognizing it as a foreign body. Differentiation into macrophages subsequently occurs followed by fusion to form foreign body giant cells (FBGCs). Consequently, implants can become degraded, cause chronic inflammation or become isolated by fibrous encapsulation. In this study, a relationship between material surface chemistry and the FBGC response was demonstrated by seeding mature monocyte‐derived macrophages (MDMs) on polycarbonate‐based polyurethanes that differed in their chemical structures (synthesized with poly(1,6‐hexyl 1,2‐ethyl carbonate) diol, and either 14C‐hexane diisocyanate and butanediol (BD) (referred to as HDI) or 4,4′‐methylene bisphenyl diisocyanate and 14C‐BD (referred to as MDI)) and material degradation assessed. At 48 h of cell‐material interaction, the FBGC attached to HDI were more multinucleated (73%) compared to MDI or the polystyrene (PS) control (21 and 36%, respectively). There was a fivefold increase in the synthesis and secretion of a protein with an approximate molecular weight of 48 kDa and a pI of 6.1 (determined by two‐dimensional gel electrophoresis) only from cells seeded on HDI. Immunoprecipitation confirmed that MSE and CE were synthesized and secreted de novo. Immunoblotting also showed an increase in secreted monocyte‐specific esterase (MSE) and cholesterol esterase (CE) from cells seeded on HDI relative to PS and MDI. Significantly more radiolabel (14C) release and esterase activity were elicited by MDMs on HDI than MDI (P < 0.05). The material that was more degradable (HDI), elicited greater protein synthesis and esterase secretion as well as more multinucleated MDMs than MDI, suggesting that the material surface chemistry modulates the function of MDM at the site of an inflammatory response to an implanted device. J. Cell. Physiol. 199: 8–19, 2004© 2003 Wiley‐Liss, Inc.
Utilizing endocrine secretory pathways in salivary glands for systemic gene therapeutics
Authors: Voutetakis Antonis , Wang Jianghua , Baum Bruce J.
Published: February 2004

Mammalian salivary glands are commonly used models of exocrine secretion. However, there is substantial experimental evidence showing the physiological existence of endocrine secretory pathways in these tissues. The use of gene transfer technology in vivo has allowed the unambiguous demonstration of these endocrine pathways. We and others have exploited such findings and evaluated salivary glands as possible target tissues for systemic applications of gene therapeutics. Salivary glands present numerous advantages for this purpose, including being well encapsulated, which limits extra‐glandular vector dissemination, and having the luminal membranes of almost all parenchymal cells accessible via intraoral delivery of vectors through the main excretory ducts. Existing studies suggest that clinical benefits will result from salivary gland targeted systemic gene therapeutics. J. Cell. Physiol. 199: 1–7, 2004. Published 2003 Wiley‐Liss, Inc.
Inosine stimulates chemotaxis, Ca2+‐transients and actin polymerization in immature human dendritic cells via a pertussis toxin‐sensitive mechanism independent of adenosine receptors
Authors: Idzko Marco , Panther Elisabeth , Bremer Hinrich C. , Windisch Wolfram , Sorichter Stephan , Herouy Yared , Elsner Peter , Mockenhaupt Maja , Girolomoni Giampiero , Norgauer Johannes
Published: February 2004

Inosine is an endogenous purine nucleoside, which is formed by adenosine deaminidase during adenosine breakdown and is released into the extracellular space from the sympathetic nervous system or injured cells. Here, we studied the biological activity of inosine on human dendritic cells (DC), which are specialized antigen presenting cells characterized by their ability to migrate from the blood to peripheral tissues, and then to secondary lymphoid organs where they initiate adaptive immune responses. In immature DC, inosine concentration‐dependently stimulated Ca2+‐transients, actin polymerization, and chemotaxis. Experiments with adenosine receptor antagonists and pertussis toxin (PTX) as well as desensitization studies suggested that the activity of inosine was mediated by a G protein‐coupled receptor pathway independent of adenosine receptors. DC, induced to mature by lipopolysaccharide, lost their ability to respond towards inosine with these activities. Moreover, inosine did neither influence membrane expression of CD54, CD80, CD83, CD86, HLA‐DR, and MHC class I molecules nor modulated secretion of interleukin (IL)‐12, IL‐10, and tumor necrosis factor alpha in immature and lipopolysaccharide‐matured DC. In aggregate, our study indicates that inosine may be involved in the trafficking control system of immature DC, and mediates its chemotactic activity by a PTX‐sensitive mechanism independent of adenosine receptors. J. Cell. Physiol. 199: 149–156, 2004© 2003 Wiley‐Liss, Inc.
Down‐regulation of MEK/ERK signaling by E‐cadherin‐dependent PI3K/Akt pathway in differentiating intestinal epithelial cells
Authors: Laprise Patrick , Langlois Marie‐Josée , Boucher Marie‐Josée , Jobin Christian , Rivard Nathalie
Published: February 2004

In vitro experiments have shown that the establishment of cell–cell contacts in intestinal epithelial cell cultures is a critical step in initiating ERK inhibition, cell cycle arrest, and induction of the differentiation process. Herein, we determined the mechanisms through which E‐cadherin‐mediated cell–cell contacts modulate the ERK pathway in intestinal epithelial cells. We report that: (1) removal of calcium from the culture medium of newly confluent Caco‐2/15 cells (30 min, 4 mM EGTA) results in the disruption of both adherens and tight junctions and clearly decreases Akt phosphorylation while increasing MEK and ERK activities. Akt, MEK, and ERK activation levels return to control levels 60 min after calcium restoration; (2) the use of E‐cadherin blocking antibodies efficiently prevents Akt phosphorylation and MEK–ERK inhibition after 70 min of calcium restoration; (3) using the PI3K inhibitor LY294002 (15 μM) in calcium switch experiments, we demonstrate that the assembly of adherens junctions activates Akt activity and triggers the inhibition of ERK1/2 activities in a PI3K‐dependent manner; (4) adenoviral infection of confluent Caco‐2/15 cells with a constitutively active mutant of Akt1 strongly represses ERK1/2 activities; (5) inhibition of PI3K abolishes Akt activity but leads to a rapid and sustained activation of the MEK–ERK1/2 in confluent differentiating Caco‐2/15 cells, but not in undifferentiated growing Caco‐2/15 cells. Our data suggest that E‐cadherin engagement leads to MEK/ERK inhibition in a PI3K/Akt‐dependent pathway. This mechanism may account for the role of E‐cadherin in proliferation/differentiation transition along the crypt‐villus axis of the human intestinal epithelium. J. Cell. Physiol. 199: 32–39, 2004© 2003 Wiley‐Liss, Inc.
Intrinsic hematopoietic stem cell/progenitor plasticity: Inversions
Authors: Colvin Gerald A. , Lambert Jean‐François , Moore Brian E. , Carlson Jane E. , Dooner Mark S. , Abedi Mehrdad , Cerny Jan , Quesenberry Peter J.
Published: February 2004

Traditional concepts indicate that stem cells give rise to progenitor cells in a hierarchical system. We studied murine engraftable stem cells (ESCs) and progenitors in in vitro and found that ESC and progenitors exist in a reversible continuum, rather then a hierarchy. B6.SJL and BALB/c marrow cells were serially cultured with thrombopoietin (TPO), FLT‐3 ligand (FLT‐3L), and steel factor through cell cycle. Progenitors (high‐proliferative potential colony‐forming cells (HPP‐CFC) and colony‐forming unit culture (CFU‐c)) and ESC capacity was determined. The cell cycle status of purified lineagenegativerhodaminelowHoechstlow stem cells was determined under the same conditions using tritiated thymidine incorporation and cell counts. We found an inverse relationship between progenitors and ESC, which occurred during the first cell cycle transit and was reversible. We have termed these progenitor/stem cell inversions and found that these inversions were consistently seen at 28–32 h of culture, representing early S‐phase. We observed 13 major reversible increases in progenitor numbers from one time‐point to another during the first cell cycle transit; this was coupled with 11 major ESC decreases and in 2 instances ESC were at baseline. These studies indicate that primitive marrow cells reversibly shift from ESC to progenitors without differentiation occurring. They exist as a fluctuating continuum. J. Cell. Physiol. 199: 20–31, 2004© 2003 Wiley‐Liss, Inc.
RACK1 is a functional target of the E1A oncoprotein
Authors: Severino Anna , Baldi Alfonso , Cottone Giuliano , Han Mei , Sang Nianli , Giordano Antonio , Maria Mileo Anna , Paggi Marco G. , De Luca Antonio
Published: February 2004

The adenoviral E1A proteins have been implicated in promotion of proliferation and transformation, inhibition of differentiation, induction of apoptosis, regulation of transcription, and suppression of tumor growth. The ability of E1A to override the fundamental controls of host cells is based on its ability to physically interact with several cellular proteins. We recently characterized RACK1 as a new E1A‐interacting protein. In this report, we show that the extreme N‐terminal region of E1A, spanning from aminoacids 1–36, and the conserved WD regions of RACK1 are responsible for this interaction. We also demonstrate that E1A and RACK1 colocalize at the perinuclear membrane in the cells. Furthermore, we provide evidence that E1A is able to antagonize the inhibitory effects of RACK1 on Src activity. These results suggest that RACK1 signaling pathway may be a functional target of E1A, contributing to E1A oncogenic effect in the host cells. J. Cell. Physiol. 199: 134–139, 2004© 2003 Wiley‐Liss, Inc.
Inhibition of ShcA isoforms p46/p52Shc enhances HIV‐1 replication in CD4+ T‐lymphocytes
Authors: Benetti Luca , Calistri Arianna , Ulivieri Cristina , Cabrelle Anna , Baldari Cosima T. , Palù Giorgio , Parolin Cristina
Published: February 2004

HIV‐1 infection decreases the number of CD4+ T‐cells, and apoptosis has been suggested among the mechanisms. Proteins of the Shc family are involved in a complex network of signal transduction, differentiation, and apoptotic response to stress in many different cell types. Out of three homologous gene products (ShcA, ShcB, and ShcC), only two splicing variants of ShA are expressed in T‐lymphocytes, namely p46Shc and p52Shc. In the present study, we report that inhibition of p46Shc and p52Shc by a dominant negative mutant enhances the yield of HIV‐1 particles production without affecting efficiency of viral gene expression in CD4+‐infected cells. The increase in HIV‐1 replication in cells expressing the dominant negative mutant isoform ultimately correlates with a decrease in the percentage of cells entering apoptosis. The data presented suggest that ShcA proteins can play a role in committing CD4+ T‐cells to apoptosis, as a response to HIV‐1 infection. J. Cell. Physiol. 199: 40–46, 2004© 2003 Wiley‐Liss, Inc.
Activity of the matrix metalloproteinase‐9 promoter in human normal and tumor cells
Authors: Morelli Cristina , Campioni Katia , Parolin Cristina , Palù Giorgio , Tognon Mauro
Published: February 2004

Matrix metalloproteinases (MMPs) belong to a family of proteins essential for those processes involving extracellular matrix degradation, such as embryonic development, morphogenesis, and tissue resorption and remodeling. Some members of this family play a crucial role also in tumor invasion. Most notably, MMP‐9 is expressed in invasive tumors, and represents a key protein in brain tumor progression, whereas it is not expressed in adult normal tissues. The expression of the MMP‐9, like other members of the family, is transcriptionally regulated. We, therefore, postulated that the MMP‐9 promoter could be useful in driving selective expression of exogenous genes in tumor cells. This represents a key feature for gene therapy applications, since currently employed viral promoters induce severe organ toxicity, limiting the clinical benefits. In this study, we investigated the activity of the MMP‐9 promoter in driving exogenous gene expression in human cell lines. High levels of reporter gene expression were detected in tumor derived cell lines, whereas the MMP‐9 promoter activity in non‐tumor cells was negligible. Furthermore, we show that tumor necrosis factor alpha (TNFα) is able to enhance considerably the MMP‐9 promoter activity only in tumor cells. Since recent studies have indicated that MMP‐9 enzymatic activity is detectable in the blood, it would be possible to screen potential responsive patients for a tumor gene therapy approach based on the MMP‐9 promoter. Taken together these data suggest that MMP‐9 promoter has the characteristics for transcritpionally targeted and inducible gene therapy applications. J. Cell. Physiol. 199: 126–133, 2004© 2003 Wiley‐Liss, Inc.
Actin re‐distribution in response to hydrogen peroxide in airway epithelial cells
Authors: Boardman Kendrick C. , Aryal Ashish M. , Miller William M. , Waters Christopher M.
Published: February 2004

Reactive oxygen species (ROS) disrupt the barrier function of airway epithelial cells through a mechanism that appears to involve remodeling of the actin cytoskeleton. Similarly, keratinocyte growth factor (KGF) has been shown to protect against ROS‐induced loss of barrier function through a mechanism that may also involve the actin cytoskeleton. To further determine the role of the actin cytoskeleton in ROS‐induced barrier injury, we quantified the relative amount of total actin associated with the cytoskeleton following exposure to hydrogen peroxide (H2O2) and pretreatment with KGF. We also determined the role of the actin–myosin contractile mechanism in the process by quantifying the relative amount of myosin heavy chain (MHC) associated with the cytoskeleton. While the transepithelial resistance (TER) of a monolayer of airway epithelial cells (Calu‐3) decreased after 2 h of continuous exposure to 0.5 mM H2O2, actin and MHC, both dissociated from the cytoskeleton within 15 min of H2O2 exposure. The TER of the monolayers remained depressed although both actin and myosin returned to the cytoskeleton by 4 h after the initiation of H2O2 exposure. Filamentous actin (f‐actin) staining suggested that the re‐associating actin took the form of short fibers associated with cortical actin rather than long stress fibers. Furthermore, pretreatment with KGF prevented the loss of actin and MHC from the actin cytoskeleton but did not prevent the decrease in TER. These studies suggest that actin disassembly from the cytoskeleton is important in the loss of barrier function, but that it is not the overall amount of actin that is associated with the cytoskeleton that is important, rather it is the contribution this actin makes to the architectural cohesiveness of the cell that contributes to the barrier function. J. Cell. Physiol. 199: 57–66, 2004© 2003 Wiley‐Liss, Inc.
Crosstalk of hypoxia‐mediated signaling pathways in upregulating plasminogen activator inhibitor‐1 expression in keloid fibroblasts
Authors: Zhang Qunzhou , Wu Yidi , Chau Cindy H. , Ann David K. , Bertolami Charles N. , Le Anh D.
Published: February 2004

Keloids are skin fibrotic conditions characterized by an excess accumulation of extracellular matrix (ECM) components secondary to trauma or surgical injuries. Previous studies have shown that plasminogen activator inhibitor‐1 (PAI‐1) can be upregulated by hypoxia and may contribute to keloid pathogenesis. In this study we investigate the signaling mechanisms involved in hypoxia‐mediated PAI‐1 expression in keloid fibroblasts. Using Northern and Western blot analysis, transient transfections, and pharmacological agents, we demonstrate that hypoxia‐induced upregulation of PAI‐1 expression is mainly controlled by hypoxia inducible factors‐1α (HIF‐1α) and that hypoxia leads to a rapid and transient activation of phosphatidylinositol‐3‐kinase/Akt (PI3‐K/Akt) and extracellular signal‐regulated kinases 1/2 (ERK1/2). Treatment of cells with PI‐3K/Akt inhibitor (LY294002) and tyrosine protein kinase inhibitor (genistein) significantly attenuated hypoxia‐induced PAI‐1 mRNA and protein expression as well as promoter activation, apparently via an inhibition of the hypoxia‐induced stabilization of HIF‐1α protein, attenuation of the steady‐state level of HIF‐1α mRNA, and its DNA‐binding activity. Even though disruption of ERK1/2 signaling pathway by PD98059 abolished hypoxia‐induced PAI‐1 promoter activation and mRNA/protein expression in keloid fibroblasts, it did not inhibit the hypoxia‐mediated stabilization of HIF‐1α protein and the steady‐state level of HIF‐1α mRNA nor its DNA binding activity. Our findings suggest that a combination of several signaling pathways, including ERK1/2, PI3‐K/Akt, and protein tyrosine kinases (PTKs), may contribute to the hypoxia‐mediated induction of PAI‐1 expression via activation of HIF‐1α in keloid fibroblasts. J. Cell. Physiol. 199: 89–97, 2004© 2003 Wiley‐Liss, Inc.
Ceramide induces caspase‐dependent and ‐independent apoptosis in A‐431 cells
Authors: Zhao Sheng , Yang Ya‐Nan , Song Jian‐Guo
Published: February 2004

We investigated the ceramide‐induced apoptosis and potential mechanism in A‐431 cells. Ceramide treatment causes the round up and the death of A‐431 cells that is associated with p38 activation and can be observed in 10 h. Short‐time ceramide treatment‐induced cell death is not associated with the typical apoptotic phenotypes, such as the translocation of phosphatidylserine (PS) from inner layer to outer layer of the plasma membrane, loss of mitochondrial membrane potential, DNA fragmentation, caspase activation, and PARP or PKC‐δ degradation. SB202190, a specific inhibitor of p38 mitogen‐activated protein (MAP) kinase, but not caspase inhibitor, blocks the cell death induced by short‐time ceramide treatment (within 12 h). Whereas neither inhibition of p38 MAP kinase nor inhibition of caspases blocks cell death induced by prolonged ceramide treatment. Moreover, incubation of cells with ceramide for a long time (over 12 h) results in the reduction of proportion of S phase accompanied with typical apoptotic cell death phenotypes that are different from the cell death induced by short‐time ceramide treatment. Our data demonstrated that ceramide‐induced apoptotic cell death involves both caspase‐dependent and caspase‐independent signaling pathways. The caspase‐independent cell death that occurred in relatively early stage of ceramide treatment is mediated via p38 MAP kinase, which can progress into a stage that is associated with changes of cell cycle events and involves both caspase‐dependent and ‐independent mechanisms. J. Cell. Physiol. 199: 47–56, 2004© 2003 Wiley‐Liss, Inc.
Latent TGF‐β1 activation by platelets
Authors: Blakytny Robert , Ludlow Anna , Martin Gail E.M. , Ireland Grenham , Lund Leif R. , Ferguson Mark W.J. , Brunner Georg
Published: February 2004

Platelets are a major source of transforming growth factor‐β1 (TGF‐β1) in the circulation as they release latent growth factor in response to activation. We report here that human platelets, when stimulated with thrombin, activated a significant proportion of the latent TGF‐β released. Latent TGF‐β activation was independent of cytokine release, since activation was delayed compared to platelet degranulation. Activation occured in releasates and did not require the continuous presence of platelets. Classical mechanisms of latent TGF‐β activation were not involved, since activation was not affected by gene deletion and/or inhibitors of the known TGF‐β activators/co‐factors, thrombospondin‐1 (TSP‐1), mannose 6‐phosphate/insulin‐like growth factor‐II receptor (M6P/IGF‐IIR), plasminogen/plasmin, or several other candidate proteases. In contrast, latent TGF‐β activation was significantly inhibited by the furin inhibitors, decanoyl‐Arg‐Val‐Lys‐Arg‐chloromethyl ketone and L‐hexaarginine. We show that platelets contain a furin‐like enzyme which is released upon platelet activation. We conclude that, following activation, platelets release and activate latent TGF‐β1 via mechanisms involving the release and activity of a furin‐like proprotein convertase. This novel mechanism of latent TGF‐β activation might represent an important mediator and therapeutic target of platelet TGF‐β1 functions, for example, in early wound repair, fibrosis, or arteriosclerosis. J. Cell. Physiol. 199: 67–76, 2004© 2003 Wiley‐Liss, Inc.
Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin‐releasing peptide's morphogenic properties
Authors: Glover Sarah , Delaney Melissa , Dematte Cecile , Kornberg Lori , Frasco Melissa , Tran‐Son‐Tay Roger , Benya Richard V.
Published: February 2004

We have proposed that gastrin‐releasing peptide (GRP) and its receptor (GRP‐R) are morphogens that when aberrantly re‐expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP‐induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non‐malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK‐related non‐kinase (FRNK) under control of a Tet‐On (i.e., doxycycline‐sensitive) promoter. Under serum‐free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP‐R antagonist D‐Phe6(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone‐plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time‐lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette <50% the size of the non‐deformed cell. Control 293 cells adhered more avidly to their underlying matrix, rapidly remodeled wounded tissues without any increase in overall proliferation, and were less distensible than cells treated with antagonist or doxycycline. Thus, these findings suggest that expression of GRP/GRPR in cancer inhibits metastasis by enhancing cell attachment to the matrix, regulating motility in the context of remodeling, and decreasing deformability. J. Cell. Physiol. 199: 77–88, 2004© 2003 Wiley‐Liss, Inc.
MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF‐2 in fibroblasts
Authors: Kesavan Kamala , Lobel‐Rice Katherine , Sun Weiyong , Lapadat Razvan , Webb Saiphone , Johnson Gary L. , Garrington Timothy P.
Published: February 2004

Mitogen‐activated protein kinases (MAPKs) are regulated by MAPK kinases (MKKs), which are in turn regulated by MKK kinases (MKKKs). While a single MKKK can regulate several different MAPK family members, and several MKKKs can often activate the same MAPK, emerging evidence indicates a unique role for individual MKKKs in acting as signaling nodes to coordinately activate different subsets of MAPKs in response to specific cellular stimuli. Thus, while there is much apparent overlap in MAPK regulation by different MKKKs, each MKKK serves a specific purpose in regulation of unique cellular functions. The purpose of this study was to define the specific role of MEKK2, an MKKK, in MAPK regulation and cell function. MEKK2 coordinately activates the ERK5 and JNK pathways. Targeted disruption of MEKK2 expression causes loss of ERK5 and JNK activation in response to FGF‐2 in mouse embryonic fibroblasts (MEFs). FGF‐2 receptor signaling requires MEKK2 for induction of mRNA for c‐Jun, Fra‐1, and Fra‐2, components of the AP‐1 transcription complex. In FGF‐2‐stimulated MEKK2−/− fibroblasts, c‐Jun phosphorylation is inhibited, consistent with a loss of JNK activation. Thus, MEKK2 regulates AP‐1 activity at two levels, by regulating both expression of AP‐1 components and c‐Jun N‐terminal phosphorylation. One function of the AP‐1 transcription complex is to regulate cytokine gene expression. Expression of IL‐1α, IL‐1β, IL‐6, and TNFα is inhibited in MEKK2−/− fibroblasts. Bacterial lipopolysaccharide (LPS) and TNFα neither activate ERK5 nor require MEKK2 for JNK activation, demonstrating specificity of MEKK2 in FGF‐2 receptor signaling and control of cytokine gene expression. J. Cell. Physiol. 199: 140–148, 2004© 2003 Wiley‐Liss, Inc.
Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cells
Authors: Xie Jiansong , Qian Limin , Wang Yanru , Rose Chadron M. , Yang Tao , Nakamura Tamako , Hamm‐Alvarez Sarah F. , Mircheff Austin K.
Published: February 2004

The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [125I]‐EGF exhibited saturable and non‐saturable, temperature‐dependent components, suggesting both receptor‐mediated and fluid phase endocytosis. Accumulation of [125I] was time‐dependent over a 120‐min period, but the content of intact [125I]‐EGF decreased after reaching a maximum at 20 min. Analytical fractionation by sorbitol density gradient centrifugation and phase partitioning indicated that within 20 min at 37°C [125I] reached an early endosome, basal–lateral recycling endosome, pre‐lysosome, and lysosome. Small components of the label also appeared to reach the Golgi complex and trans‐Golgi network. Intact [125I]‐EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [125I]‐labeled degradation products appeared in the pre‐lysosomes and lysosomes. Confocal microscopy imaging of FITC‐EGF and LysoTrackerRed revealed FITC enriched in a dispersed system of non‐acidic compartments at 20 min and in acidic compartments at 120 min. Both confocal immunofluorescence microscopy and analytical fractionation indicated that the intracellular EGFR pool was much larger than the plasma membrane‐expressed pool at all times. Cells loaded with [125I]‐EGF released a mixture of intact EGF and [125I]‐labeled degradation products. The observations indicate that in lacrimal acinar cells, EGFR and EGF–EGFR complexes continually traffic between the plasma membranes and a system of endomembrane compartments; EGF‐stimulation generates time‐dependent signals that initially decrease, then increase, EGF–EGFR traffic to degradative compartments. J. Cell. Physiol. 199: 108–125, 2004© 2003 Wiley‐Liss, Inc.
8‐prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo
Authors: Pepper Michael S. , Hazel Susan J. , Hümpel Michael , Schleuning Wolf‐Dieter
Published: February 2004

8‐Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three‐dimensional collagen gel within which they form capillary‐like tubes, we demonstrate that 8‐prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC50 of between 3 and 10 μM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8‐prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8‐prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co‐added. These findings suggest that 8‐prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component. J. Cell. Physiol. 199: 98–107, 2004© 2003 Wiley‐Liss, Inc.
Effects of inorganic anions on photoactivity of various photocatalysts under different conditions
Authors: Hu Chun , Yuchao Tang , Lanyu Lin , Zhengping Hao , Yizhong Wang , Hongxiao Tang
Published: February 2004

The effects of inorganic anions (SO42−, H2PO4−, HCO3−, F−) on the photoactivity of surface bond‐conjugated TiO2/SiO2 and Fe–TiO2/SiO2 for the photodegradation of Acid Red B were investigated. The introduction of iron changed the chemical environment of lattice titanium and the surface charge properties of TiO2/SiO2. Fe–TiO2/SiO2 inhibited the negative role of the selected inorganic anions. The effects of different light sources and dyes of various structure, with a sulfonate group, on the role of F− in decolorization of Acid Red B were examined. F− has the same negative influence on the photoactivity of TiO2/SiO2 with the blacklight lamp and solar irradiation, but shows no influence upon irradiation with the 254 nm lamp. The tested inorganic anions affected the photodegradation of dyes by controlling their adsorption onto the surface of the catalyst and by trapping h+ and •OH. Copyright © 2004 Society of Chemical Industry
The use of 3,3′,4′,5‐tetrachlorosalicylanilide as a chemical uncoupler to reduce activated sludge yield
Authors: Chen Ying Xu , Ye Fen Xia , Feng Xiao Shan
Published: February 2004

To determine whether chemical additions can be used to reduce sludge production in biological wastewater treatment, 3,3′,4′,5‐tetrachlorosalicylanilide (TCS) was added to activated sludge cultures as a metabolic uncoupler. Batch tests confirmed that TCS is an effective chemical uncoupler in reducing the sludge yield at concentrations greater than 1.0 mg dm−3; a TCS concentration of 1.0 mg dm−3 reduced sludge yield by approximately 50%. Substrate removal capability and effluent nitrogen concentration were not affected adversely by the presence of TCS when dosed every other day in a range of 2.0–3.6 mg dm−3 during the 40‐day operation of activated sludge batch cultures. Such sludge growth reduction was associated with the enhancement of microbial activities in terms of the specific oxygen uptake rate and dehydrogenase activity. Sludge settleability of the treated and control samples was qualitatively comparable and not significantly different. Filamentous bacteria continued to grow in sludge flocs only in the control reactor at the end of the 40‐day trial. These results suggest that TCS treatment of activated sludge systems may reduce excess sludge yield. Copyright © 2003 Society of Chemical Industry
Phenol degradation in a fixed‐bed bioreactor using micro‐cellular polymer‐immobilized Pseudomonas syringae
Authors: Erhan E , Yer E , Akay G , Keskinler B , Keskinler D
Published: February 2004

Highly porous (85% void volume) polymer beads with interconnecting micro‐pores were prepared for the immobilization of Pseudomonas syringae for the degradation of phenol in a fixed‐bed column bioreactor. The internal architecture of this support material (also known as PolyHIPE Polymer) could be controlled through processing before the polymerization stage. The transient and steady state phenol utilization rates were measured as a function of substrate solution flow rate and initial substrate concentration. The spatial concentration of the bacteria on the micro‐porous support particles as well as within them was studied using scanning electron microscopy at various time intervals during the continuous operation of the bioreactor. It was found that although bacterial penetration into the porous support was present after 20 days, bacterial viability however, was compromised after 120 days as a result of the formation of a biofilm on the support particles. The steady state phenol utilization at an initial phenol concentration of 200 mg cm−3 was 100% provided that the flow rate was less than 7 cm3 min−1. Substrate inhibition at a constant flow rate of 4.5 cm3 min−1 was found to begin at 720 mg dm−3. The critical dilution rate for bacteria washout was high as a result of the highly hydrophobic nature of the support and the reduction of pore interconnect size due to bacterial growth within the pores in the vicinity of the surface of the support. Copyright © 2004 Society of Chemical Industry