bluuebunny/crossref_metadata_2025 huggingface dataset:
Asymmetric Direct Aldol Reaction Catalysed by L‐Prolinethioamides
Authors: Gryko Dorota , Lipiński Radosław
Published: June 2006

L‐Prolinethioamides have been found to be active catalysts for direct aldol reactions of acetone with aromatic aldehydes, affording aldol products in good yields and with good enantioselectivities. They were prepared from L‐proline and simple aliphatic and aromatic amines in optically pure form and in good overall yields. Studies employing ten catalysts allowed us to unequivocally establish the basic principles governing the outcome of the L‐prolinethioamide‐catalysed aldol reaction. In particular, the catalyst prepared from L‐proline and (S)‐phenylethylamine catalysed the reaction of acetone with 4‐cyanobenzaldehyde in 57 % yield and 93 % ee (100 % ee at –78 °C). Most importantly, we found that steric interaction between the catalyst and a donor or an acceptor is crucial for the stereoselectivity of the aldol addition, while the unwanted formation of imidazolidinethione (from the catalyst and acetone or an aldehyde) was shown to decrease both the ee and the yield. The influence of the amine moiety (–CSNHR), different solvents and temperatures were studied, and we also found that there is a linear correlation between the optical purity of the catalyst and the ee of the aldol product, which supports the hypothesis that the reaction proceeds by the enamine–imine mechanism, involving only one molecule of the catalyst in the transition state. For the first time, the formation of 1,5‐dihydroxypentan‐3‐one products (double addition products) was studied in detail. By precise optimisation we were able to show that the courses of the reactions of acetone with highly reactive aromatic aldehydes could be manipulated to give either the aldol products or 1,5‐dihydroxypentan‐3‐one derivatives as the major product in moderate ee. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
Distribution of low‐density lipoprotein‐bound low‐molecular‐weight thiols: A new analytical approach
Authors: Zinellu Angelo , Sotgia Salvatore , Zinellu Elisabetta , Formato Marilena , Manca Salvatorica , Magliona Stefania , Ginanneschi Renzo , Deiana Luca , Carru Ciriaco
Published: June 2006

We have recently demonstrated that low‐density lipoprotein (LDL) apoprotein is able to bind the most concentrated plasma thiols such as cysteine, cysteinylglycine, and homocysteine by disulfide linkage. However, the LIF CE assay employed to measure linked thiols was not sensitive enough to verify whether low concentrated plasma thiols as glutathione and glutamylcysteine are also linked to apoprotein. By modifying sample treatment and electrophoretic parameters we set up a new method with an LOQ of about 1.5 nmol/L, by which we demonstrate that LDL apoprotein binds all physiological plasma thiols. The increased sensitivity was obtained by drying released apoB thiols after reduction treatment, dissolving them directly in a low volume of derivatization buffer and decreasing the dilution factor of derivatized sample before CE injection. Moreover, by increasing the concentration of the electrolyte buffer, we improved the selectivity of peaks, in particular between glutathione (GSH) and the impurity peak derived from unreacted 5‐iodoacetamidofluorescein, which in the previous electrophoretic conditions were overlapped. The method optimization, reached by searching the best combination between sample matrix and CE run buffer, is fully described. Given the potential pathologic significance of protein thiolation, the proposed method may be useful to understand the mechanisms and the balances that regulate the interaction between thiols and –SH free groups of proteins.
Comparison of oligonucleotide migration in a bicontinuous cubic phase of monoolein and water and in a fibrous agarose hydrogel
Authors: Sanandaji Nima , Carlsson Nils , Voinova Marina , Åkerman Björn
Published: June 2006

Porous hydrogels such as agarose are commonly used to analyze DNA and water‐soluble proteins by electrophoresis. More recently lyotropic liquid crystals, such as the diamond cubic phase formed by the lipid monoolein and water, has become a new type of well‐defined porous structure of interest for both hydrophilic and amphiphilic analytes. Here we compare these two types of matrixes by investigating the nature of retardation they confer to an oligonucleotide that migrates in their respective aqueous phases. The retardation for a 25‐mer oligonucleotide was found to be about 35‐fold stronger in the cubic phase than in an agarose hydrogel modified to have the same average pore size. According to modelling, the strong retardation is primarily due to the fact that hydrodynamic interaction with the continuous monoolein membrane is a stronger source of friction than the steric interactions (collisions) with discrete gel fibres. A secondary effect is that the regular liquid crystal has a narrower pore‐size distribution than the random network of the agarose gel. In agreement with experiments, these two effects together predict that the retardation in the cubic phase is a 30‐fold stronger than in an agarose gel with the same average pore radius.
On‐chip chiral separation based on bovine serum albumin‐conjugated carbon nanotubes as stationary phase in a microchannel
Authors: Weng Xuexiang , Bi Hongyan , Liu Baohong , Kong Jilie
Published: June 2006

A novel method of chiral separation based on protein‐stationary phase immobilized in a poly(methyl methacrylate) microfluidic chip was developed. BSA conjugated with the shortened carboxylic single‐walled carbon nanotubes (SWNTs) was employed as the chiral selector. Successful separation of tryptophan enantiomers was achieved in less than 70 s with a resolution factor of 1.35 utilizing a separation length of 32 mm. This is the first example of chiral separation based on SWNTs‐BSA conjugates as stationary phase immobilized in microchip channel. The stability of the stationary phase in the channel was examined by microchip electrophoresis with laser‐induced fluorescence detection. Factors that influenced the chiral separation resolution were examined. Under the optimized conditions, the proposed modified chip revealed adequate repeatability concerning run‐to‐run. These results show that the use of SWNTs‐BSA conjugates within microfluidic channels hold great promise for a variety of analytical schemes.
A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2‐DE and MS analysis
Authors: Song Jun , Braun Gordon , Bevis Eric , Doncaster Kristen
Published: June 2006

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol‐free protein extraction procedures for 2‐DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS‐PAGE and 2‐DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2‐DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2‐DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.
Knowledge‐based proteome profiling: Considering identified proteins to evaluate separation efficiency by 2‐D PAGE
Authors: Gundacker Nina , Bayer Editha , Traxler Elisabeth , Zwickl Hannes , Kubicek Markus , Stöckl Johannes , Gerner Christopher
Published: June 2006

Proteome profiling techniques rely on the separation of proteins or peptides and their subsequent quantification. The reliability of this technique is still limited because a proteome profiling result does not necessarily represent the true protein composition of the analysed sample, thus seriously hampering proper data interpretation. Many experimentally observed proteome alterations are biologically not significant. It was the aim of this study to use the knowledge of the biological context of proteins in order to establish optimised proteome profiling protocols. While 2‐D spot patterns of total cell protein fractions were found to poorly represent the true protein composition, purified subcellular protein fractions were found to better represent the protein composition of the analysed sample. The application of a standardised protocol to different kinds of cells revealed several striking observations. Firstly, the protein composition of cultured cells of various origins is very similar. Secondly, proteome alterations observed with the described protocols do make sense from a biologic point of view and may thus be considered as truly representative for the analysed samples. Thirdly, primary white blood cells isolated from different donors were found to show minor, but reproducible and significant individual differences. We designate the consideration of known properties of identified proteins in proteome profiles as a knowledge‐based approach. The present data suggest that this approach may tremendously help to improve the applied techniques and assess the results. We demonstrate that the fulfilment of well‐defined criteria of proteome profiles eventually results in reliable and biologically relevant data.
Binding of Ca2+, Mg2+, and heparin by human serum amyloid P component in affinity capillary electrophoresis
Authors: Heegaard Niels H. H. , He Xinya , Blomberg Lars G.
Published: June 2006

Human serum amyloid P component (SAP) is a glycoprotein circulating in the blood and found in association with all types of amyloid (malfolded potein aggregates) examined so far. Despite uncertainties regarding the precise function of SAP in vivo, the lectin‐like properties of this Ca2+‐activated protein with affinity for anionic saccharides and malfolded proteins are well known. The propensity to form homomeric penta‐ or decamers in solution and the selfaggregation in the presence of Ca2+ as well as the tendency of SAP to attach to uncoated fused silica have precluded the analysis of SAP by microelectrophoretic methods. We now work out conditions to characterize the binding of Ca2+ and Mg2+ and the binding of heparin to SAP in the presence of divalent metal ions by ACE. The results show a strong binding of heparin (sub‐μM apparent dissociation constants) even in the abscence of Ca2+ at low ionic strength, pH 8.2. Also, a selective interaction with Ca2+ compared with Mg2+ is demonstrated. The approach will further the use of microelectrophoretic methods to examine the interactions of SAP with ligands of putative pathophysiological relevance such as lipopolysaccharides and misfolded proteins.
Detection and analysis of protein–protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes
Authors: Krause Frank
Published: July 2006

It is an essential and challenging task to unravel protein–protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome‐wide scale in a single experiment. This review focus on blue‐native (BN)‐PAGE as the most versatile and successful gel‐based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN‐PAGE is a charge‐shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless‐native (CN)‐PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein–protein interactions better than BN‐PAGE. The essential conditions determining the success of detecting protein–protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN‐ and CN‐PAGE studies to elucidate protein–protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN‐ and CN‐PAGE.
Expression and function of calcium‐activated potassium channels in human glioma cells
Authors: Weaver Amy K. , Bomben Valerie C. , Sontheimer Harald
Published: June 2006

Ca2+‐activated K+ (KCa) channels are a unique family of ion channels because they are capable of directly communicating calcium signals to changes in cell membrane potential required for cellular processes including but not limited to cellular proliferation and migration. It is now possible to distinguish three families of KCa channels based on differences in their biophysical and pharmacological properties as well as genomic sequence. Using a combination of biochemical, molecular, and biophysical approaches, we show that human tumor cells of astrocytic origin, i.e. glioma cells, express transcripts for all three family members of KCa channels including BK, IK, and all three SK channel types (SK1, SK2, and SK3). The use of selective pharmacological inhibitors shows prominent expression of currents that are inhibited by the BK channel specific inhibitors iberiotoxin and paxilline. However, despite the presence of transcripts for IK and SK, neither clotrimazole, an inhibitor of IK channels, nor apamin, known to block most SK channels inhibited any current. The exclusive expression of functional BK channels was further substantiated by shRNA knockdown experiments, which selectively reduced iberiotoxin sensitive currents. Western blotting of patient biopsies with antibodies specific for all three KCa channel types further substantiated the exclusive expression of BK type KCa channels in vivo. This finding is in sharp contrast to other cancers that express primarily IK channels. © 2006 Wiley‐Liss, Inc.
Improving irrigated agriculture performance through an understanding of the water delivery process
Authors: Clemmens Albert J.
Published: June 2006

The performance of large‐scale irrigation projects worldwide has been disappointing to the international community. Continued poor performance could limit our ability to provide food and fibre for a growing, more affluent world population. Improvement in the productivity of large irrigation systems is a key component to assuring future adequate food and fibre supplies. This paper discusses the reasons for poor performance of these schemes and proposes a method to improve their performance. A main problem is that operation of these irrigation systems is not tied to productivity. As a result, the dispersive nature of these large open canal distribution systems causes extreme variability in water delivery service to users. The remedy is to break the system down at key intermediate locations within the network and to improve physical and administrative control at those locations. Published in 2006 by John Wiley & Sons, Ltd.
Impacts of drainage activities for clay soils on hydrology and solute loads to surface water
Authors: van den Eertwegh G. A. P. H. , Nieber J. L. , de Louw P. G. B. , van Hardeveld H. A. , Bakkum R.
Published: July 2006

Several extended field‐monitoring programmes were set up in the Netherlands to study hydrological and surface water quality impacts of agricultural drainage activities for clay soils.

At the field scale, agricultural land use data and hydrological measurements were combined with analysis of drainage water quality data, and related to the loading of the surface water system with salts and nutrients. Water, salt, and nutrient balances of the soil and/or surface water system were set up to analyse the hydrological behaviour of the systems studied and to quantify solute sources.

Field observations were performed by field data analysis and numerical modelling, using the 1D F1W‐MultiSWAP code, a 2D dual‐porosity code, and the MODFLOW code. Drainage activities showed a significant impact on hydrology by influencing subsurface drainage water flow paths and regional‐scale groundwater seepage.

Through solute balances it was shown that the salt and nutrient loading of the surface water were clearly affected by both agricultural and drainage activities. The conclusion was that the drainage activities and their surface water quality aspects need to be studied in an integrated way. Changes in subsurface drainage system redesign, surface water level management, and the field‐scale nutrient management need to be integrated. Copyright © 2006 John Wiley & Sons, Ltd.
Global gene expression analysis of the effects of vinblastine on endothelial cells, when eluted from a thermo‐responsive polymer
Authors: McLucas E. , Gallagher H. , Rochev Y. , Carroll W.M. , Gorelov A. , Smith T.J.
Published: June 2006

In‐stent restenosis remains a significant problem associated with bare metal stents. This drawback has prompted research into improving stent design and the development of novel coatings, including drug‐eluting stents. A number of drug‐eluting stents are currently on the market; however, the success rate of these stents in complex situations has been found to be quite low. Thus, there remains potential for the development of more suitable drug‐eluting stents. The aims of this study were to use a thermoresponsive polymer to develop a system to locally deliver vinblastine, an antimitotic agent currently used as an anticancer drug, and in addition, assess the effects of this drug at the gene expression level in vitro. An N‐isopropylacrylamide/N‐tert‐butylacrylamide (NiPAAm/NtBAAm) copolymer solution in the ratio 65:35 was prepared and appropriate volumes of vinblastine were added to generate two final drug concentrations of 22 nanomoles/film or 0.022 nanomoles/film. Stainless steel discs (316) were coated with the copolymer solution or this solution containing drug. Human endothelial cells were cultured on collagen type 1 gels and then incubated with the coated discs for 24 h. Gene expression studies using oligonucleotide microarray analysis and quantitative RT‐PCR were then performed. Microarray analysis revealed that vinblastine causes the differential expression of a range of genes involved in a variety of different functions, including cell cycle and apoptosis. The changes in expression of some of these genes culminate in cell cycle arrest and apoptotic pathways. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Biomineralization of polyanionic collagen–elastin matrices during cavarial bone repair
Authors: Rocha Lenaldo B. , Adam Randall L. , Leite Neucimar J. , Metze Konradin , Rossi Marcos A.
Published: June 2006

The polyanionic collagen–elastin matrices (PCEMs) are osteoconductive scaffolds that present high biocompatibility and efficacy in the regeneration of bone defects. In this study, the objective was to determine if these matrices are directly mineralized during the osteogenesis process and their influence in the organization of the new bone extracellular matrix. Samples of three PCEMs, differing in their charge density, were implanted into critical‐sized calvarial bone defects created in rats and evaluated from 3days up to 1 year after implantation. The implanted PCEMs were directly biomineralized by osteoblasts as shown by ultrastructural, histoenzymologic, and morphologic analysis. The removal of the implants occurred during the bone remodeling process. The organization of the new bone matrix was evaluated by image texture analysis determining the Shannon's entropy and the fractal dimension of digital images. The bone matrix complexity decreased as the osteogenesis progressed approaching the values obtained for the original bone structure. These results show that the PCEMs allow faster formation of new bone by direct biomineralization of its structure and skipping the biomaterial resorption phase. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Substrate mineralization stimulates focal adhesion contact redistribution and cell motility of bone marrow stromal cells
Authors: Leonova Elena V. , Pennington Keith E. , Krebsbach Paul H. , Kohn David H.
Published: June 2006

Understanding the mechanisms of substrate based control of cell function is critical to the design of biomaterials. Cells interact with their extracellular matrix through cell adhesion contacts. We have previously described the self assembly of bone‐like mineral onto an organic template and have shown that these biomimetic surfaces lead to an increased volume fraction of bone regenerated in vivo. In the present study, we compared the distribution of cell adhesion contacts, cell spreading, and cell motility of murine bone marrow stromal cells (BMSC) on mineralized vs. nonmineralized substrates. We developed a new approach for quantification of cell‐material interactions and demonstrated that cell adhesion contacts on mineralized substrates were distributed throughout the cell surface contacting the substrate, whereas on nonmineralized substrates cell adhesion contacts were present near the cell periphery. We propose that mineralized substrates stimulate the predominant expression of fibrillar contacts, and nonmineralized substrates stimulate expression of focal adhesion contacts. Cell motility assays with colloidal gold demonstrated a statistically significant decrease in the average phagokinetic index of migrating cells on mineralized vs. nonmineralized substrates after 90 min of cell seeding. We propose that the physical–chemical properties of the substrate, altered by mineralization, cause expression of specific types of cell contacts and, as a result, modify molecular mechanisms responsible for cell spreading, motility, and possibly differentiation. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Adsorption of bisphosphonate onto hydroxyapatite using a novel co‐precipitation technique for bone growth enhancement
Authors: McLeod Kate , Anderson Gail I. , Dutta Naba K. , Smart Roger St.C. , Voelcker Nicolas H. , Sekel Ron , Kumar Sunil
Published: June 2006

Premature bone resorption and remodeling by osteoclasts can limit the longevity of implant fixation and recovery time. Orally administered bisphosphonates (BPs) have been used to inhibit osteoclast action at the implant/bone interface. Ideally, these should be delivered at the interface with the osteoblast‐active hydroxyapatite (HA) for maximum effect. This investigation introduces a novel BP loading technique to achieve improved BP release from a simulated body fluid‐grown HA (SBF‐HA) with the aim of improving implant fixation. A solution co‐precipitation technique incorporates the BP (pamidronate) into a thin SBF‐HA coating. Surface analysis, using X‐ray photoelectron spectroscopy (XPS), of the resultant coating was employed to confirm the presence of the adsorbed BP on the surface of SBF‐HA. XPS analysis was also used to determine the optimal adsorption process. Osteoclast cell culture experiments confirmed the biological effectiveness of BP adsorption and proved that the pamidronate was biologically active, causing both decreased osteoclast numbers and decreased resorption. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Strain patterns during tensile, compressive, and shear fatigue of human cortical bone and implications for bone biomechanics
Authors: Winwood K. , Zioupos P. , Currey J.D. , Cotton J.R. , Taylor M.
Published: June 2006

It is a common theme in basic bone biomechanics and in biomechanical applications that much of the behavior can be determined and is dictated by the level of strain, whether this pertains to bone physiology, bone remodeling, osseoinduction, osseointegration, or the development of damage. The development of damage, demonstrated by stiffness loss measurements, has already been reported in detail in the literature. However, the systematic study of the development of “plastic” (residual) strains, which are associated with the inelastic mechanical behavior of bone tissue, has generally been overlooked. The present study compares the rates at which the elastic (ea) and plastic components (ep) of strain developed during tensile, compressive, and shear fatigue in human cortical bone of six individuals aged between 53 and 79 years. The overall hypothesis of this investigation is that there is a common underlying factor in the damage‐related behavior of bone, which may allow us to link together the various aspects of the damage related behavior of bone. The rate of development of plastic strain (Δep/ΔN) and the rate of growth in elastic strain amplitude (Δea/ΔN) are described as a function of the stress (σ), and/or stress normalized by the modulus of elasticity (σ/E). The implications of our findings are discussed with respect to simple models/mechanisms, which may underlie the observed behavior. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Effects of interconnecting porous structure of hydroxyapatite ceramics on interface between grafted tendon and ceramics
Authors: Omae Hiromichi , Mochizuki Yu , Yokoya Shin , Adachi Nobuo , Ochi Mitsuo
Published: June 2006

The purpose of this study was to evaluate histologically and biomechanically the interface between porous hydroxyapatite ceramics and a tendon grafted into ceramics, and to compare the interface in two ceramics with different porous structures: interconnected porous calcium hydroxyapatite ceramics (IP‐CHA) with an effective porosity index (interpore diameter > 20 μm) of 63.6%, and porous calcium hydroxyapatite ceramics with less interconnection (HA‐L) with an effective porosity index of 5.5%. The tendon‐IP‐CHA complex and the tendon‐HA‐L complex were implanted into the bone defects made in both knees of rabbits. With IP‐CHA, abundant fibrous tissue, including vessels and collagen fiber continuity, was observed inside interface‐region pores. The amount of osseous tissue in interface‐region pores increased over time, and at 24 weeks after operation, the tendon was in direct contact with the osseous tissue in IP‐CHA. With HA‐L, the amount of fibrous tissue in interface‐region pores was low and did not increase. The results of biomechanical analysis revealed that the maximum tendon pull‐out load from IP‐CHA was significantly higher than that from HA‐L. With the porous hydroxyapatite ceramics having highly interconnecting porous structure, a bioactive interface was achieved between ceramics and grafted tendon. On the basis of these results, we conclude that bone defects, including tendon insertion, can be reconstructed using IP‐CHA. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Mineralization of hydroxyapatite in electrospun nanofibrous poly(L‐lactic acid) scaffolds
Authors: Chen Jinglu , Chu Benjamin , Hsiao Benjamin S.
Published: June 2006

A highly porous electrospun poly(L‐lactic acid) (PLLA) nanofibrous scaffold was used as a matrix for mineralization of hydroxyapatite. The mineralization process could be initiated by immersing the electrospun scaffold in the simulated body fluids (SBF) at 37°C for varying periods of time. Scanning electron microscopy (SEM), energy‐dispersive spectroscopy (EDS), wide‐angle X‐ray diffraction (WAXD), Fourier transform infrared (FTIR), and Raman spectroscopy were used to characterize the composition and the structure of the deposited mineral on the nanofiber surface. Results indicated that the mineral phase was a carbonated apatite with thin flake‐like nanostructures. The effects of functional groups on the scaffold surface and anionic additives in the incubation fluids on the nucleation and growth of the mineral were investigated. It was found that a minuscule amount of anionic additives (e.g., citric acid and poly‐L‐aspartic acid) in the SBF could effectively inhibit the mineral growth. Surface modification of the scaffold was carried out by hydrolysis of PLLA scaffold in NaOH aqueous solution, where carboxylic acid groups were produced without compromising the scaffold integrity. The mineralization process from modified PLLA electrospun scaffolds was significantly enhanced because the calcium ions can bind to the carboxylate groups on the fiber surface. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Effects of biomaterial surface chemistry on the adhesion and biofilm formation of Staphylococcus epidermidis in vitro
Authors: MacKintosh Erin E. , Patel Jasmine D. , Marchant Roger E. , Anderson James M.
Published: June 2006

The formation of biofilm, a structured community of bacteria enclosed in slime, is a significant virulence factor in medical‐device‐centered infection. The development of cardiovascular device infection can be separated into two phases: initial bacterial adhesion and aggregation, followed by proliferation and production of slime. It is possible to modulate the adhesion and biofilm formation of Staphylococcus epidermidis, a commensal skin bacterium commonly found on infected medical devices, through biomaterial surface chemistry. This study examines bacterial adhesion and biofilm formation on surface‐modified polyethylene terephthalate (PET), including surfaces with varying hydrophilic, hydrophobic, and ionic character. Bacterial adhesion and biofilm formation were observed over 48 hours in phosphate‐buffered saline (PBS) and 20% pooled human serum. The hydrophilic surface (PAAm) had significantly less nonspecific adhesion of bacteria than that in the control (PET) and other surfaces, when cultured in PBS (P < 0.0001). Charged surfaces, both anionic and cationic, had increased adhesion and aggregation of bacteria in comparison with the control (PET) in the presence of serum proteins over 24 hours (P < 0.0001). Bacteria cultured in serum on the charged surfaces did not have significantly different amounts of biofilm formation compared with that of the control (PET) surface after 48 hours. This study showed that biomaterial surface chemistry characteristics impact initial adhesion and aggregation of S. epidermidis on biomaterials. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Overexpression of regucalcin enhances glucose utilization and lipid production in cloned rat hepatoma H4‐II‐E cells: Involvement of insulin resistance
Authors: Nakashima Chikage , Yamaguchi Masayoshi
Published: July 2006

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of glucose utilization and lipid production was investigated using the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin. The hepatoma cells (wild‐type) and stable regucalcin/pCXN2‐transfected cells (transfectant) were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. Cells with subconfluency were cultured for 24 or 72 h in medium containing either vehicle or insulin (10−8 or 10−7 M) with or without supplementation of glucose (10, 25, or 50 mg/ml of medium) in the absence of insulin. The production of triglyceride and free fatty acid was significantly increased in transfectants cultured without insulin and glucose supplementation as compared with that of wild‐type cells. The supplementation of glucose (10, 25, or 50 mg/ml) caused a remarkable increase in medium glucose consumption, triglyceride, and free fatty acid productions in transfectants cultured without insulin. The presence of insulin (10−7 M) caused a significant increase in medium glucose consumption, triglyceride, and free fatty acid productions in wild‐type cells cultured with glucose supplementation. These increases were significantly prevented in transfectants cultured for 72 h. The expression of acetyl‐CoA carboxylase, HMG‐CoA reductase, glucokinase, pyruvate kinase, and glyceroaldehyde‐3‐phosphate dehydrogenase (G3PDH) mRNAs in wild‐type cells was not significantly changed by culture with or without glucose supplementation in the presence of insulin. These gene expressions were not significantly changed in transfectants. The expression of glucose transporter 2 mRNA was significantly increased in transfectants as compared with that of wild‐type cells. Such an increase was not seen in transfectants cultured in the presence of insulin with or without glucose supplementation. This study demonstrates that overexpression of regucalcin enhances glucose utilization and lipid production in the cloned rat hepatoma H4‐II‐E cells, and that it regulates the effect of insulin. J. Cell. Biochem. 99: 1582–1592, 2006. © 2006 Wiley‐Liss, Inc.
Calcium‐pH Crosstalks in the human mast cell line HMC‐1: Intracellular alkalinization activates calcium extrusion through the plasma membrane Ca2+‐ATPase
Authors: Pernas‐Sueiras Octavio , Alfonso Amparo , Vieytes Mercedes R. , Orfao Alberto , Escribano Luis , Francisca Sánchez‐Jiménez , Botana Luis M.
Published: July 2006

The human mast cell line (HMC‐1) has been used to study the relationship between intracellular pH and cytosolic calcium (Ca2+) in mast cells. Thapsigargin (TG) caused store‐operated Ca2+ entry, that is enhanced by the PKC activator PMA. NH4Cl‐induced alkalinization showed an inhibitory effect on TG‐sensitive stores depletion (not on TG‐insensitive stores), and also on final cytosolic Ca2+ levels reached in response to both TG and the ionophore ionomycin. Loperamide, a positive modulator of store‐operated channels, induced a slight Ca2+ entry by itself, and also increased TG‐induced Ca2+ entry. This enhancement was not enough to reverse the inhibitory effect of NH4Cl‐induced alkalinization. When comparing the effect of NH4Cl‐induced alkalinization on Ca2+ levels, with those observed using Ca2+ channel blockers (namely Ni2+ and SKF‐96365), cytosolic profiles for this ion are different, either in modified saline solution or in HCO‐free medium. Thus, it seems unlikely that the inhibitory effect of NH4Cl‐induced alkalinization on Ca2+ is taking place by blockage of Ca2+ entry. Furthermore, inhibition of the plasma membrane Ca2+‐ATPase (an important mechanism for Ca2+ efflux) with sodium orthovanadate (SO) matches with the inhibition of the negative effect on Ca2+ levels elicited by NH4Cl. Data indicate that NH4Cl‐induced alkalinization might be activating Ca2+ efflux from the cell, by stimulation of the plasma membrane Ca2+‐ATPase, and also confirm our previous finding that Ca2+ is a secondary signal to activate HMC‐1 cells. J. Cell. Biochem. 99: 1397–1408, 2006. © 2006 Wiley‐Liss, Inc.
Transcriptional regulation of the homeobox gene NKX3.1 by all‐trans retinoic acid in prostate cancer cells
Authors: Thomas Marc A. , Hodgson Myles C. , Loermans Susan D. , Hooper Joel , Endersby Raelene , Bentel Jacqueline M.
Published: July 2006

NKX3.1 is a homeobox gene, expression of which is largely restricted to the adult prostatic epithelium. Loss of NKX3.1 expression has been linked to prostate carcinogenesis and disease progression and occurs in the absence of mutations in the coding region of the NKX3.1 gene. In this study, we have characterized regulation of NKX3.1 expression by all‐trans retinoic acid (tRA), a naturally occurring vitamin A metabolite that is accumulated at high levels in the prostate. Using the prostate cancer cell line LNCaP, Western blot analysis revealed a ∼twofold induction of NKX3.1 protein levels following tRA exposure, with sequential analysis of NKX3.1 protein levels in cycloheximide co‐treated cells indicating that tRA does not alter NKX3.1 protein turnover. The ∼1.6‐fold increase in NKX3.1 mRNA levels detected in tRA‐treated LNCaP cells also occurred independently of new protein synthesis and was not mediated by changes in NKX3.1 mRNA stability. In contrast, nuclear run‐on assays indicated that tRA treatment increased NKX3.1 transcription. To identify retinoid responsive regions of the NKX3.1 gene, DNA sequences encompassing ∼2 kb of the NKX3.1 promoter or the entire 3′untranslated region (UTR) were cloned into luciferase reporter plasmids. Analysis of induced luciferase activity following transfection of these constructs into prostate cancer cells did not identify tRA responsiveness, however the 3′UTR was found to be strongly androgen responsive. These studies demonstrate that the NKX3.1 gene is a direct target of retinoid receptors and suggest that androgen regulation of NKX3.1 expression is mediated in part by the 3′UTR. J. Cell. Biochem. 99: 1409–1419, 2006. © 2006 Wiley‐Liss, Inc.
Involvement of caspase activation and mitochondrial stress in trichostatin A‐induced apoptosis of Burkitt's lymphoma cell line, Akata
Authors: Son Young‐Ok , Choi Ki‐Choon , Lee Jeong‐Chae , Kook Sung‐Ho , Lee Hyun‐Jeong , Jeon Young‐Mi , Kim Jong‐Ghee , Kim Ju , Lee Won‐Keun , Jang Yong‐Suk
Published: July 2006

Epstein–Barr virus (EBV) infects more than 90% of the human population and has a potential oncogenic nature. Trichostatin A (TSA) has potent antitumor activity, but its exact mechanism on EBV‐infected cells is unclear. This study examined the effects of TSA on proliferation and apoptosis of the Burkitt's lymphoma cell line, Akata. TSA treatment inhibited cell growth and induced cytotoxicity in both the EBV‐negative and ‐positive Akata cells. TSA sensitively induced apoptosis in both cells, as demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to sub‐G1 phase by flow cytometric analysis, and the formation of DNA ladders. This suggests that EBV has no effect on the sensitivity to TSA. Western blot analysis showed that the cleavage of PARP and Bid and the activation of caspases are closely related to the TSA‐induced apoptosis of the cells. The reduction in mitochondrial transition potential and the release of apoptosis‐inducing factor from mitochondria to cytosol was also observed after the TSA treatment, but was suppressed by treating the cells with a cathepsin B inhibitor. Overall, these findings suggest that besides the caspase‐dependent pathway, mitochondrial events are also associated with the TSA‐induced apoptosis of Akata cells. J. Cell. Biochem. 99: 1420–1430, 2006. © 2006 Wiley‐Liss, Inc.
Leading a horse to water: An action perspective on mental health policy
Authors: Davidson Larry , Flanagan Elizabeth , Roe David , Styron Thomas
Published: June 2006

Following publication of several landmark policy documents beginning in 1999, there are unprecedented opportunities for reforming mental health care to meet the needs of persons with mental illness. In this article, the authors suggest that transforming mental health requires dramatic changes in theory as well as in policy and practice, offering action theory as a corrective for a clinical psychology that has yet to view people as active agents shaping their own lives. A participatory approach to policy development and adoption of an action‐oriented model of clinical practice provide examples of the shift, which results from treating people to enhancing their access to opportunities and offering them the in vivo supports they need to pursue meaningful lives even while disabled. © 2006 Wiley Periodicals, Inc. J Clin Psychol 62: 1141–1155, 2006.
A mass spectrometric investigation on the possible role of tryptophan and 7‐hydroxytryptophan in melanogenesis
Authors: Smaniotto Anna , Comai Stefano , Bertazzo Antonella , Costa Carlo V. L. , Allegri Graziella , Seraglia Roberta , Traldi Pietro
Published: June 2006

The activity of tyrosinase and peroxidase + H2O2 in promoting melanogenesis from tryptophan (Trp) and 7‐hydroxytryptophan (7‐HTP) has been investigated. The reaction samples have been drawn at different reaction times and analysed by MALDI mass spectrometry.

The data obtained showed that tryptophan undergoes, under tyrosinase and peroxidase action, an oligomerization process mainly due to the reaction of anthranilic acid (AA) and Trp. However, analysing the UV and fluorescence data, it is seen that the oligomers cannot belong to the melanin pattern, but their possible role in melanogenesis is not to be excluded. Once it reacts with the two enzymes, 7‐hydroxytryptophan leads to dark brown products, indicating its possible role in melanin production. In contrast to what was observed in the case of 5‐hydroxytryptophan, for which oligomers were constituted by 5‐hydroxytryptophan (5‐HTP) and 5‐hydroxytryptamine (5‐HT) units, the MALDI data indicate a sharply different behaviour for 7‐HTP. In fact, in the case of 5‐hydroxytryptophan, oligomerization takes place through the formation of 5‐hydroxytryptamine and the oligomerization products are due to mixed 5‐HTP—5‐HT oligomers. In the case of 7‐hydroxytryptophan, the formation of 7‐hydroxytryptamine (7‐HT) is also observed, but it does not seem to play any role; the only oligomerization products formed are due to the reaction of 7‐hydroxytryptophan and AA. The data so obtained indicate that 7‐hydroxytryptophan acts like an effective melanin precursor in the presence of both tyrosinase and peroxidase + H2O2. Copyright © 2006 John Wiley & Sons, Ltd.
C‐terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C‐terminus
Authors: Li Zhong , Yalcin Talat , Cassady Carolyn J.
Published: June 2006

Deprotonated peptides containing C‐terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off‐resonance irradiation collision‐induced dissociation (SORI‐CID) in a Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix‐assisted laser desorption ionization/time‐of‐flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI‐CID and PSD, [M − H]− and [M − 2H]2− underwent an unusual cleavage corresponding to the elimination of the C‐terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C‐terminus or the side chain carboxylate group of C‐terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C‐terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C‐terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C‐terminus, C‐terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C‐terminal residue elimination in the negative ion mode. Copyright © 2006 John Wiley & Sons, Ltd.
A new strategy utilizing electrospray ionization‐quadrupole ion trap mass spectrometry for the qualitative determination of GnRH peptides
Authors: Myers Tanya R. , Patonay Gabor
Published: June 2006

Numerous forms of the neurotransmitter GnRH have been discovered in vertebrates and invertebrates. Methods used for identification of these peptides are laborious and often require the application of multiple, confirmatory techniques. In this study, we investigate whether HPLC‐MS/MS and de novo sequencing techniques applied to whole peptide analysis can provide a simpler approach to GnRH characterization. Experiments were performed with six GnRH forms (chicken I, chicken II, lamprey III, mammalian, salmon and seabream) to determine whether MS/MS spectra would be dominated by proline‐directed fragmentation to the detriment of obtaining sufficient fragmentation for sequencing. While the expected b8 fragment was prominent, sufficient ion series were obtained for the six GnRH peptides to provide sequence identification. On the basis of the patterns observed for six model peptides, similar fragmentation patterns are expected for other GnRH forms. To confirm the applicability of the method, extracts from Sprague–Dawley rat brains were examined. These experiments confirm the presence of mammalian GnRH and a posttranslationally modified form of mammalian GnRH, hydroxyproline9 GnRH, in Sprague–Dawley rat brains and demonstrate that ESI‐MS/MS techniques provide a valuable addition to existing qualitative methods. Copyright © 2006 John Wiley & Sons, Ltd.
On the determination of molecular weight distributions of asphaltenes and their aggregates in laser desorption ionization experiments
Authors: Hortal Ana R. , Martínez‐Haya Bruno , Lobato Marcos D. , Pedrosa José M. , Lago Santiago
Published: June 2006

Molecular weight distributions (MWD) of asphaltenes and their aggregates have been investigated in laser desorption ionization (LDI) mass spectrometric experiments. A systematic investigation of the dependence of the measured MWD on the asphaltene sample density and on the laser pulse energy allows the assignment of most probable molecular weights within 300–500 amu and average molecular weights of 800–1000 amu for the monomeric asphaltenes, as well as for the estimation of the contribution from asphaltene clusters in typical LDI measurements. The results serve to reconcile the existing controversy between earlier mass spectrometric characterizations of asphaltenes based on laser desorption techniques by different groups. Furthermore, the MWD measurements performed on particularly dense samples yield an additional differentiated broad band peaking around 9000–10 000 amu and extending over 20 000 amu, not observed previously in LDI experiments, thereby revealing a strong propensity of the asphaltenes to form clusters with specific aggregation numbers, which is in qualitative agreement with previous theoretical predictions and with the interpretation of measurements performed with other techniques. Copyright © 2006 John Wiley & Sons, Ltd.
Alfred Nier and the sector field mass spectrometer
Authors: De Laeter John , Kurz Mark D.
Published: June 2006

Science and technology are intimately related, and advances in science often become possible with the availability of new instrumentation. This has certainly been the case in mass spectrometry, which is used in so many scientific disciplines. Originally developed as an instrument for research in physics it was used in the discovery of isotopes, their recognition as the fundamental species comprising the elements, and the investigation of elemental isotopic composition. Isotope ratio mass spectrometry is a metrological technique of the highest order, and has been widely used in chemical, biochemical, cosmochemical, environmental, geological, physical, and nuclear research. Mass spectrometry presently plays a key role not only in scientific research, but also in industrial operations.

This paper highlights the role that Alfred Otto Carl Nier played in bringing mass spectrometry into the mainstream of science. Nier's career spanned a remarkable period in science, and he made crucial contributions to atomic weights, geochronology, isotope geochemistry, nuclear physics, and space science. He is widely viewed as the ‘father of modern mass spectrometry’, because of his genius with instrumentation, his innovations, and the generosity with which he shared his ideas and designs. It is timely to remember his fundamental work in mass spectrometry, particularly the development of the sector field mass spectrometer, which is still the instrument of choice for many isotope scientists some 66 years after its first appearance in 1940. Copyright © 2006 John Wiley & Sons, Ltd.