bluuebunny/crossref_metadata_2025 huggingface dataset:
Shallow site archaeology: Artifact dispersal, stratigraphy, and radiocarbon dating at the Barger Gulch locality B Folsom Site, Middle Park, Colorado
Authors: Surovell Todd A. , Waguespack Nicole M. , Mayer James H. , Kornfeld Marcel , Frison George C.
Published: June 2005

Shallowly buried archaeological sites are particularly susceptible to surface and subsurface disturbance processes. Yet, because cultural deposition often operates on short time scales relative to geologic deposition, vertical artifact distributions can be used to clarify questions of site formation. In particular, patterns in artifact distributions that cannot be explained by occupation histories must be explained by natural processes that have affected sites. Buried only 10–50 cm beneath the ground surface for 10,450 14C yr, the Folsom component at Barger Gulch Locality B (Middle Park, Colorado) exhibits many signs of post‐depositional disturbance. Through examination of variation in the vertical distribution of the artifact assemblage, we are able to establish that only a Folsom component is present. Using vertical artifact distributions, stratigraphy, and radiocarbon dating, we are able to reconstruct the series of events that have impacted the site. The Folsom occupation (˜10,450 14C yr B.P.) was likely initially buried in a late‐Pleistocene eolian silt loam. Erosion brought the artifacts to rest on a deflation surface at some time prior to 9400 14C yr B.P. A mollic epipedon formed in sediments that accumulated between 9400 and 7000 14C yr B.P. Some time after 5200 14C yr B.P., this soil was partially truncated, and artifacts that had previously dispersed upward created a secondary lag at its upper contact. This surface was buried again and artifact dispersal continued. © 2005 Wiley Periodicals, Inc.
In vitro studies of calcium phosphate glass ceramics with different solubility with the use of human bone marrow cells
Authors: Dias A. G. , Lopes M. A. , Trigo Cabral A. T. , Santos J. D. , Fernandes M. H.
Published: June 2005

Two glass ceramics in the CaOP2O5MgO system with the incorporation of K2O or TiO2 oxides were prepared with the goal of using them as potential bone graft substitutes. The incorporation of TiO2 and K2O led to the preparation of specific crystalline phases in the structure of the glass ceramics, which show different degrees of biodegradation. In fact, the 45CaO45P2O55MgO5K2O has been previously demonstrated to be much more soluble in aqueous solutions than the 45CaO37P2O55MgO13TiO2 glass ceramic. The in vitro biological activity of the two calcium phosphate glass ceramics was studied with the use of human bone marrow osteoblast cell cultures maintained for 28 days, and seeded materials were assessed for cell proliferation and function. The Ti‐containing glass ceramic showed a stable surface throughout the culture time, on macroscopic and SEM observation. Osteoblast cells proliferated gradually, especially during the third week, with a high alkaline phosphatase activity and formation of a mineralized matrix. On SEM observation, attached cells appeared with a spread‐polygonal morphology typical of the osteoblast cells, with extensive cell‐to‐cell contact. Cell behavior on the seeded material was similar to that found on cultures performed on tissue‐culture‐grade polystyrene; except for the presence of lower cell numbers during the first 2 weeks. By contrast, the K‐containing glass ceramic showed a highly instable surface with dissolution/precipitation processes occurring throughout the culture time. Few cells adhered to the material surface, and subsequent proliferation was also hindered, especially from the first week onwards. Cell numbers were significantly lower than those observed in the Ti‐containing glass ceramic during most of the incubation time. Results suggest that the different in vitro biological behavior of these two glass ceramics is mainly due to the significant differences in the surface degradation rate, which is directly correlated to the chemical composition of the mother glass. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005
Porous scaffolds of gelatin–hydroxyapatite nanocomposites obtained by biomimetic approach: Characterization and antibiotic drug release
Authors: Kim Hae‐Won , Knowles Jonathan C. , Kim Hyoun‐Ee
Published: June 2005

Gelatin–hydroxyapatite (HA) nanocomposite porous scaffolds were fabricated biomimetically, and their feasibility as a drug‐delivery carrier for tissue‐regeneration and wound‐healing treatments was addressed. The composite sols were prepared by the precipitation of HA up to 30 wt % within a gelatin solution with the use of calcium and phosphate precursors, and the porous scaffold was obtained by casting the sols and further freeze drying. The obtained bodies were crosslinked with carbodiimide derivatives to retain chemical and thermal integrity. The apatite precipitates were observed to be a poorly crystallized carbonate‐substituted HA. The nanocomposite scaffolds had porosities of ∼ 89–92% and exhibited a bimodal pore distribution, that is, the macropores (∼ 300–500 μm) of the framework structure, and micropores (∼ 0.5–1 μm) formed on the framework surface. Transmission electron microscopy (TEM) observation revealed the precipitation of highly elongated HA nanocrystals on the gelatin network. The well‐developed porous structure and organized nanocomposite configurations were in marked contrast to the directly mixed gelatin–HA powder conventional composites. For drug‐release tests, tetracycline, an antibiotic drug, was entrapped within the scaffold, and the drug‐release profile was examined with processing parameters, such as HA amount in gelatin, crosslinking degree, and initial drug addition. The drug entrapment decreased with increasing HA amount, but increased with increasing crosslinking degree and initial drug addition. The crosslinking of the gelatin was the prerequisite to sustaining and controlling the drug releases. Compared to pure gelatin, the gelatin–HA nanocomposites had lower drug releases, because of their lower water uptake and degradation. All the nanocomposite scaffolds released drugs in proportion to the initial drug addition, suggesting their capacity to deliver drugs in a controlled manner. Based on the findings of the well‐developed morphological feature and controlled drug‐release profile, the gelatin–HA nanocomposite porous scaffolds are suggested to be potentially useful for hard‐tissue regeneration. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2005
Angiogenesis, endothelial cell functions, and tumor cell growth in biodegradable and nonbiodegradable devices
Authors: Zhao Xia , Kalliomäki Suzanne , Wang Xiangdong , Kalliomäki Jacob , Andersson Roland
Published: June 2005

Development of tissue engineering creates multiple potentials for clinical treatment and scientific research. Biodegradable collagen matrices have been found to support simultaneous autotransplantation of hepatocytes after major liver resection. Dynamic angiogenesis in biodegradable devices (BDD) and nondegradable devices (NDD) transplanted into the renal subcapsule and subcutaneous tissue was measured by the distribution of radiolabeled red blood cells and serum albumin. The circulation, microvascular integrity, and capacities of endothelial cells (adhesion, proliferation, and migration) were investigated within 2 weeks after subcutaneous transplantation of both devices. Patterns of tumor cell growth in both devices were morphologically studied. After subcutaneous transplantation, significant angiogenesis was noted at 1 week in BDD implants and from 2 weeks and on in NDD implants, with an increase in implant blood and plasma volumes. Leakage index of radiolabeled albumin in NDD implants was significantly higher than in BDD implants, while the leakage index 2 weeks after BDD implant was similar to that in subcutaneous tissues. Adhesion, proliferation and migration rates of endothelial cells isolated from both devices were higher than from subcutaneous tissues. Endothelial proliferation and migration rates in BDD implants were significantly higher at 1 week, while in NDD at 2 weeks. Tumor cells migrated and grew on the top surface of NDD with a flattened shape, while growing within the BDD forming a round mass. Endothelial capacities, angiogenetic procedure, and biological and physical characteristics of the device contribute to patterns of tumor cell growth in the device. Biodegradable collagen matrix with three‐dimensional structure is suitable for simultaneous transplantation with cells without prevascularization. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2005
Simultaneous activation of JAK1 and JAK2 confers IL‐3 independent growth on Ba/F3 pro‐B cells
Authors: Huang Huei‐Mei , Lin Yi‐Li , Chen Cheng‐Hsien , Chang Ting‐Wei
Published: June 2005

JAK1 and JAK2 are tyrosine kinases involved in the regulation of cell proliferation, differentiation, and survival. These proteins may play a key role in mediating the effects of the cytokine IL‐3 on hematopoietic cells. IL‐3 induces tyrosine phosphorylation of both JAK1 and JAK2. However, it is not clear whether the activation of JAK1, JAK2, or both is sufficient to confer factor‐independent growth in IL‐3 dependent cells. To address this issue, fusion proteins CD16/CD7/JAK (CDJAK), comprised of a CD16 extracellular domain, a CD7 transmembrane domain, and a JAK cytoplasmic region (either a wild‐type JAK or a dominant negative mutant of JAK) were constructed. We established several Ba/F3 derivatives that stably overexpress the conditionally active forms of either CDJAK1, CDJAK2, or both these fusion proteins. In this study, the autophosphorylation of CDJAK1 or CDJAK2 was induced by crosslinking with anti‐CD16 antibody. We demonstrated that, like their wild‐type counterparts, CDJAK1 and CDJAK2 were preassociated with the IL‐3 receptor beta and alpha subunits, respectively. Furthermore, the simultaneous activation of both CDJAK1 and CDJAK2 fusion proteins, but not either one alone, led to the tyrosine phosphorylation of the IL‐3 receptor beta subunit, the activation of downstream signaling molecules, including STAT5, Akt, and MAPK, and the conferring of factor‐independent growth to IL‐3‐dependent Ba/F3 cells. Coexpression of dominant negative mutants CDJAK1KE or CDJAK2KE with wild type CDJAK2 or CDJAK1, respectively, inhibited these activation activities. These results suggest that JAK1 and JAK2 must work cooperatively and not independently and that their actions are dependent on having normal kinase activity to trigger downstream signals leading to IL‐3 independent proliferation and survival of Ba/F3 cells. © 2005 Wiley‐Liss, Inc.
A paradigm for the treatment of prostate cancer bone metastases based on an understanding of tumor cell–microenvironment interactions
Authors: Loberg Robert D. , Gayed Bishoy A. , Olson Karin B. , Pienta Kenneth J.
Published: June 2005

The pliability of cancer cells to mutate into several different phenotypes in an attempt to find one that will survive and colonize at the metastatic site is a tremendous “hurdle” to overcome in designing novel cancer therapeutics. New targets of therapy are essential if we are to effectively overcome the evasiveness of cancer. The interaction between the tumor cell and the surrounding microenvironment creates a vicious cycle that perpetuates disease survival and progression. The future of cancer therapy resides in the ability to focus on the recruited and exploited relationships of the cancer cell with the host environment. These therapies target cancer cell growth early and interrupt the vicious cycle that is created by the tumor cells interacting with bone components by inhibiting osteoclasts, osteoblasts, stromal cells, and endothelial cells. They alter the bone microenvironment, creating a hostile “soil” that prevents the “seed” from developing into bone metastases and represent a potential new platform for the development of prostate cancer therapeutics. © 2005 Wiley‐Liss, Inc.
Solute transport in non‐aqueous nanofiltration: effect of membrane material
Authors: Geens Jeroen , Hillen An , Bettens Ben , Van der Bruggen Bart , Vandecasteele Carlo
Published: June 2005

Nanofiltration experiments in methanol and ethanol were carried out for six reference components with different molecular weights (MW 228–880) and polarities (logP 0–12). The contribution of diffusion to solute transport, calculations based on results from cell diffusion experiments, was found to be only 1–7%; solute transport occurs mainly by convection. Furthermore, it was found that solute transport is influenced by solute–solvent–membrane interactions. Solvent–solute interactions (solvation) cause a different effective solute diameter in each solvent: it is smaller in ethanol than in methanol, resulting in lower rejections in ethanol than in methanol. Solute rejection increases with increasing molecular size (for components with similar polarity). Solute–membrane interactions were expressed in polarity terms and charge effects. A decrease of the rejection with decreasing solute polarity (for components with similar MW) was observed. Since non‐polar components (high logP) are exposed to smaller repulsion forces from the polymeric membrane material, the resistance against solute permeation is lower for these components. The solvent–membrane interactions were found to result in solvation of the pore wall; the degree of membrane solvation is different for each solvent. It is determined by the affinity between the solvent and the membrane, and by the molecular size of the solvent. In ethanol, hydrophilic membranes show a larger drop in solute rejection than hydrophobic membranes. The differences in solvent–membrane affinity (measured by contact angle) are much smaller for the first membranes, and therefore pore wall solvation decreases with increasing solvent size. Hydrophobic membranes have a much larger affinity for ethanol than for methanol, leading to stronger interactions, but undergo competitive forces due to the larger solvent size. Therefore, the difference in degree of solvation and effective pore diameter is less pronounced. Based on these three observed or postulated interactions, rejections of all six reference solutes in methanol and ethanol could be explained. Copyright © 2005 Society of Chemical Industry
Screen‐printed biosensor for allergens
Authors: Sarkar P , Ghosh D , Bhattacharya D , Kataky R , Setford SJ , White SF , Turner APF
Published: June 2005

Allergen levels in indoor environments, leading to many diseases, eg asthma, rhinitis and conjunctivitis, affect a large and increasing fraction of the population. A quite effective and inexpensive method of a rough but very rapid overall assessment of total allergen level in the environment has been developed. The method involved estimation of protein in allergen extracts by screen‐printed electrodes using two different techniques. The biosensor comprised a rhodinised carbon working electrode, a silver/silver chloride reference electrode and a carbon counter electrode. In the first method the enzyme protease reacted with allergen protein to release amino acid, which produced hydrogen peroxide in the presence of amino acid oxidase. This was detected amperometrically. The second method used potassium bromide as electrolyte and the electrode was subjected to dual potential. Bromine, released due to electrolysis at higher potential, was consumed by the allergen protein at lower potential. In the first method, a unique technique was used to microencapsulate the enzyme protease and immobilise it on the surface of the electrode by in‐situ polymerisation to avoid contact with the amino acid oxidase. A total of seven allergens were tested and the results gave a good correlation with the standard protein measurement method. Environmental specimens from indoors, schools and workplaces can be evaluated for the aeroallergens produced by dust mites, animal hairs, cockroach debris, pollens, etc as a means of determining the exposure risk. Copyright © 2005 Society of Chemical Industry
Hyperbranched polymers generated from oxyanionic vinyl polymerization of commercially available methacrylate inimers
Authors: Jia Zhifeng , Yan Deyue
Published: June 2005

Hyperbranched polymethacrylates were prepared by means of oxyanionic vinyl polymerization of commercially available monomers, including hydroxyethyl methacrylate (HEMA) and poly(ethylene glycol) methacrylate (PEG‐MA). Hyperbranched polymethacrylates with high molecular weight were obtained with the complex of potassium hydride and 18‐crown‐6 as the initiator. The effect of 18‐crown‐6 is very important, and only oligomer can be obtained in the polymerization without 18‐crown‐6. The molecular structure of the hyperbranched polymers was confirmed with 1H NMR and 13C NMR spectra. The ratio of initiator to monomer significantly affects the architecture of the resultant polymers. When the ratio of initiator to monomer equals 1 in the oxyanionic vinyl polymerization of HEMA, the degree of branching of the resulting polymer was calculated to be around 0.49. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3502–3509, 2005
Hydrogen bonding in peptide secondary structures
Authors: Varga Zoltán , Kovács Attila
Published: June 2005

Hydrogen bonding interactions in various peptide secondary structures (β‐sheet, 27‐ribbon, 310‐helix, α‐helix, π‐helix, β‐turn II, and γ‐turn) have been investigated in small oligopeptides by quantum chemical calculations at the B3LYP/6‐31G** level. Besides the primary O…HN interactions, the optimized structures revealed the importance of N…HN hydrogen bonding in several structures. The effect of substitution on the energy and structural properties was investigated comparing the properties of glycine, alanine, valine, and serine. The aliphatic substituents generally weaken the hydrogen bonds, the strongest effects being observed in crowded valine conformers. Additional hydrogen bonding interactions introduced by the OH group of serine can both strengthen (by polarizing the amide moiety through N…H interaction) and weaken (constraining the CO oxygen by O…HO interaction) the backbone hydrogen bonds. The effect of water as a polarizable medium on the energy properties was assessed by the COSMO model. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
An evaluation of the utility of in vacuo methylation for mass‐spectrometry‐based analyses of peptides
Authors: Laremore Tatiana N. , Weber David M. , Choma Christin T.
Published: June 2005

In vacuo trimethylation of the N‐terminus of a lyophilized peptide with methyl iodide was previously reported to enhance the peptide's signal in matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) and to suppress alkali adduct formation in electrospray ionization mass spectrometry (ESI‐MS). Both the signal enhancement and alkali adduct suppression observed for methylated peptides are believed to be due to the permanent positive charge on the N‐terminus of the peptide resulting from the formation of a quaternary ammonium moiety. The present work evaluates the general utility of the in vacuo methylation procedure for the MS analysis of peptides, and specifically addresses the issue of whether the methylation of nucleophilic sites other than the N‐terminal amine affects the MALDI signal of modified peptides. This work establishes that, although certain side‐chain modifications are inevitable using present reaction conditions, the derivatization leads to significant MALDI‐MS signal improvement. The experimental results demonstrate that the N‐terminal trimethylammonium derivatives of peptides exhibit MALDI signals comparable to or exceeding those of arginine‐containing standards such as angiotensin I. The advantages and limitations of the in vacuo methylation procedure are discussed. Copyright © 2005 John Wiley & Sons, Ltd.
Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry
Authors: Stevens Stanley M. , Chung Alfred Y. , Chow Marjorie C. , McClung Scott H. , Strachan Camille N. , Harmon Alice C. , Denslow Nancy D. , Prokai Laszlo
Published: June 2005

A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta‐elimination and Michael addition chemistries in a ‘one‐step’ reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution‐ or gel‐based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low‐abundance post‐labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation‐site mapping has been evaluated for a phosphoprotein standard, bovine beta‐casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT‐labeled proteins or peptides. Copyright © 2005 John Wiley & Sons, Ltd.
Determination of proanthocyanidins in fresh grapes and grape products using liquid chromatography with mass spectrometric detection
Authors: Wu Qingli , Wang Mingfu , Simon James E.
Published: June 2005

Fresh grapes and grape products, such as grape wine and grape juice, were analyzed for proanthocyanidins (PACs) using liquid chromatography with electrospray ionization mass spectrometric (MS) detection. PACs were successfully separated and analyzed on the basis of their protonated molecules, allowing the identification of PACs in different degrees of polymerization from monomers to oligomers (up to 7 units), and in various isomeric forms. Using reversed‐phase high‐performance liquid chromatography (HPLC) combined with MS detection, the PAC monomers, (+)‐catechin (C), (−)‐epicatechin (EC), (−)‐catechin gallate (CG), and (−)‐epicatechin gallate (ECG), were successfully quantified using selected ion monitoring (SIM) mode. Standard curves were fitted for each PAC ranging from 43.8 to 5600 ng/mL for C, from 42.2 to 5400 ng/mL for EC, from 36.7 to 4700 ng/mL for CG, and from 39.8 to 5100 ng/mL for ECG. Good linearity (r2 > 0.999) was achieved for each analyte. The accuracy and precision (RSD) were within 10% (n = 8) at the limit of detection. This method allows direct quantification of monomeric PACs in fresh grapes and grape‐derived products. Additionally, flow injection analysis (FIA) was applied to estimate the concentration levels of PAC oligomers by comparing their FIA‐MS peak areas, which were well correlated (r2 = 0.936) to the total concentrations of PAC monomers. Copyright © 2005 John Wiley & Sons, Ltd.
Development of a multiresidue method for analysis of major Fusarium mycotoxins in corn meal using liquid chromatography/tandem mass spectrometry
Authors: Cavaliere Chiara , Foglia Patrizia , Pastorini Elisabetta , Samperi Roberto , Laganà Aldo
Published: June 2005

A sensitive and reliable liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) method has been developed to determine, in a single run, eight trichothecenes, three fumonisins, zearalenone and α‐zearalenol, in corn meal samples. LC and MS conditions were varied to find the best compromise in terms of sensitivity and separation. An acceptable compromise was obtained using a C18 column thermostatted at 45°C and a mobile phase gradient of methanol/water with 10 mmol/L formate buffer (pH 3.8). A multiple reaction monitoring program, in which fumonisins and trichothecenes (except nivalenol and deoxynivalenol) are acquired in positive ESI as [M+H]+ or [M+NH4]+, and all other compounds in negative ESI, was developed to match appropriate retention time windows. Sample preparation used a simple homogenization of the corn meal sample with acetonitrile/water (75:25, v/v) followed by extraction on a C18 cartridge and clean‐up on a cartridge containing graphitized carbon black. Method detection limits were in the range 2–14 ng/g, with the exception of nivalenol (27 ng/g), deoxynivalenol (40 ng/g) and 15‐acetyldeoxynivalenol (30 ng/g). Good accuracy (recoveries 81–104%) and precision (RSD 4–11%) were obtained by performing calibration using a spiked analyte‐free extract. Copyright © 2005 John Wiley & Sons, Ltd.
Proteomics of gluten: mapping of the 1Bx7 glutenin subunit in Chinese Spring cultivar by matrix‐assisted laser desorption/ionization
Authors: Alberghina Gaetano , Cozzolino Rosaria , Fisichella Salvatore , Garozzo Domenico , Savarino Anna
Published: June 2005

The verification of the cDNA‐deduced sequence of the high molecular weight glutenin subunit 1Bx7 in Chinese Spring cultivar was achieved by direct matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) analysis of the tryptic fragments. The published sequence of the 1Bx7 subunit contains 5 Lys and 15 Arg residues but, due to the presence of three Arg–Pro bonds, which are generally resistant to cleavage by trypsin, or cleaved to a very limited extent by trypsin, 19 peptides can be predicted. The identification of the tryptic fragments was achieved by direct MALDI‐MS analysis by using three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures in order to obtain the maximum sequence coverage. MALDI analysis of the 1Bx7 tryptic digest resulted in the identification of the expected peptides and additional fragments arising from non‐specific cleavages; the fragments that were not detected are peptides with low mass (from 147.2 to 317.4), so we obtained a sequence coverage of 98.8%. The results reported here also indicated that the sequence of the 1Bx7 subunit from cv. Chinese Spring is different from the cDNA‐deduced sequence reported in the literature; in particular, a possible insertion of the hexapeptide QPGQGQ within the sequence Gln630‐Tyr725 was suggested. Finally, it is possible to rule out glycosylation of the 1Bx7 subunit, or any other post‐translational modification, to within the detection limits of the method. Copyright © 2005 John Wiley & Sons, Ltd.
Structural characterization of 2‐aminobenzamide‐derivatized oligosaccharides using a matrix‐assisted laser desorption/ionization two‐stage time‐of‐flight tandem mass spectrometer
Authors: Morelle Willy , Slomianny Marie‐Christine , Diemer Hélène , Schaeffer Christine , Dorsselaer Alain van , Michalski Jean‐Claude
Published: June 2005

Oligosaccharides were derivatized by reductive amination using 2‐aminobenzamide (2‐AB) and analyzed by matrix‐assisted laser desorption/ionization two‐stage time‐of‐flight (MALDI‐TOF/TOF) tandem mass spectrometry (MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2‐AB‐derivatized oligosaccharides. A systematic study was conducted on a series of 2‐AB‐derivatized oligosaccharides to allow rationalization of the fragmentation processes. The MALDI‐TOF/TOF‐MS/MS spectra of the [M+Na]+ ions of 2‐AB‐derivatized oligosaccharides were dominated by glycosidic cleavages. These fragments originating both from the reducing and the non‐reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, the MALDI‐TOF/TOF‐MS/MS spectra were also characterized by abundant cross‐ring fragments which are very informative on the linkages of the monosaccharide residues constituting these oligosaccharides. MALDI‐TOF/TOF‐MS/MS analysis of 2‐AB‐derivatized oligosaccharides, by providing structural information at the low‐picomole level, appears to be a powerful tool for carbohydrate structural analysis. Copyright © 2005 John Wiley & Sons, Ltd.
Development of a low volume plasma sample precipitation procedure for liquid chromatography/tandem mass spectrometry assays used for drug discovery applications
Authors: Xu Xiaoying , Zhou Qiao , Korfmacher Walter A.
Published: June 2005

The demand for high sensitivity bioanalytical methods has dramatically increased in the drug discovery stage; in addition, there has been a growing trend of reducing the sample volume that is required for these assays. A sensitive high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay requires only a low plasma sample volume (10 µL) and employs a protein precipitation procedure using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient based on a methanol/water mobile phase with a flow rate set to 0.8 mL/min was used. The test method showed very good linearity between 0.1–1000 ng/mL (R2 = 0.9737), precision (%RSD = 6–9), accuracy (%RE = −2) and reproducibility (%RSD = 11). A drug discovery IV/PO study was assayed using both the new low volume method and our standard volume (50 µL) method. The correlation of the two sets of data from the two methods was excellent (R2 = 0.9287). This new assay procedure has been successfully used in our laboratory for over 100 different rat or mouse discovery PK studies. Copyright © 2005 John Wiley & Sons, Ltd.
AR4-2J cells: A model to study polypeptide hormone receptors
Authors: Dietrich Jean-Bernard
Published: June 2005

The AR4-2J cell line is derived from a transplantable tumour of the exocrine rat pancreas. Acinar in origin, this cell line contains significant amounts of amylase and can be grown in continuous culture. Many in vitro studies have been done using these cells; these studies were often complemented with in vivo experiments on animals. Particularly, many polypeptide hormones interacting with specific receptors located on the cell membrane have been analysed. The accurate knowledge of the hormone-receptor interactions has allowed to design interesting analogs of these hormones. In several cases, these compounds are powerful antagonists and are able to control cell proliferation induced by the corresponding polypeptide hormones. Other cell lines are useful to understand human pancreatic cancer. These human cell lines (Capan-1, Panc-1 for example) are of ductal origin and differ from AR4-2J cells, especially regarding the distribution of several polypeptide hormone and growth factor receptors. Both models are important for basic studies of neuropeptides, gastrointestinal peptides and their receptors, as well as for a better understanding of the underlying mechanisms of human pancreatic cancer.
Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B in associated with distinct changes in F-actin and ZO-1
Authors: Nybom Pia , Magnusson Karl-Eric
Published: June 2005

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 μM) and higher concentrations (1.5 μM and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 μg/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.
Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase
Authors: Fan G. F. , Yang X. Y. , Huang Y. G. , Yang F. Y.
Published: June 2005

In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of β-AR and adenylyl cyclase, β-AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2− gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated β-AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of β-AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled β-AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted β-AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated β-AR via Gs.
Ipsilateral Breast Tumor Recurrence after Breast‐conserving Therapy: A Comparison of Quadrantectomy versus Lumpectomy at a Single Institution
Authors: Noh Woo Chul , Paik Nam Sun , Kim Min Suk , Yang Kwang Mo , Cho Chul Koo , Choi Dong Wook , Lee Jong Inn , Kang Sung Ku , Kim Sang Bum , Moon Nan Mo
Published: June 2005

The aims of study were to compare the rates of ipsilateral breast tumor recurrence (IBTR), for patients treated with either quadrantectomy or lumpectomy at a single institution, and to identify predictors of IBTR after breast‐conserving therapy (BCT). The database and medical records of 807 patients who underwent BCT for breast cancer between 1987 and 2002 were reviewed. The age of the patient, tumor size, lymph node status, extensive intraductal component (EIC), re‐excision, final margin status, and the extent of surgery were examined in reference to IBTR rates. Of the total 807 patients, 456 (56.5%) had undergone quadrantectomy and 351 patients (43.5%) had lumpectomy. Apart from the higher re‐excision rate in the lumpectomy group (p < 0.001), there were no significant differences in clinical and pathologic characteristics between the patients in the two groups. At the median follow‐up time of 72 months, 28 cases of IBTR (3.4%) and 56 cases of systemic recurrence (6.9%) had developed in 72 patients (8.9%). On multivariate analysis, young age (≤ 35) (p = 0.041), positive lymph node (p <0.001), and the presence of EIC (p = 0.004) were independent predictors of IBTR. However, we could not find a significant difference in IBTR rate between the two groups (p = 0.546). Thus, the extent of breast surgery (quadrantectomy or lumpectomy) did not make a significant difference in IBTR if adequate surgical margins could be achieved.
Estimating hydrodynamic roughness in a wave‐dominated environment with a high‐resolution acoustic Doppler profiler
Authors: Lacy Jessica R. , Sherwood Christopher R. , Wilson Douglas J. , Chisholm Thomas A. , Gelfenbaum Guy R.
Published: June 2005

Hydrodynamic roughness is a critical parameter for characterizing bottom drag in boundary layers, and it varies both spatially and temporally due to variation in grain size, bedforms, and saltating sediment. In this paper we investigate temporal variability in hydrodynamic roughness using velocity profiles in the bottom boundary layer measured with a high‐resolution acoustic Doppler profiler (PCADP). The data were collected on the ebb‐tidal delta off Grays Harbor, Washington, in a mean water depth of 9 m. Significant wave height ranged from 0.5 to 3 m. Bottom roughness has rarely been determined from hydrodynamic measurements under conditions such as these, where energetic waves and medium‐to‐fine sand produce small bedforms. Friction velocity due to current u*c and apparent bottom roughness z0a were determined from the PCADP burst mean velocity profiles using the law of the wall. Bottom roughness kB was estimated by applying the Grant‐Madsen model for wave‐current interaction iteratively until the model u*c converged with values determined from the data. The resulting kB values ranged over 3 orders of magnitude (10−1 to 10−4 m) and varied inversely with wave orbital diameter. This range of kB influences predicted bottom shear stress considerably, suggesting that the use of time‐varying bottom roughness could significantly improve the accuracy of sediment transport models. Bedform height was estimated from kB and is consistent with both ripple heights predicted by empirical models and bedforms in sonar images collected during the experiment.
Formation of oriented thaw lakes by thaw slumping
Authors: Pelletier Jon D.
Published: June 2005

In the classic model for oriented thaw lakes, sublittoral shelves form by wind‐driven circulation near shorelines oriented perpendicular to the wind, protecting the adjacent banks from thaw and wave cut erosion. Here I propose an alternative model based on thaw slumping and test the model predictions against observations in northern Alaska. Thermal modeling illustrates that bank height controls the rate of thaw slumping because summertime thaw penetrates only decimeters into a tall bank but as much as tens of meters into a short bank. This effect also leads to oriented lakes because of a nonlinear relationship between bank height and bank retreat rate. Bank material texture also controls the rate of thaw slumping because fine‐grained sediments drain slowly and maintain higher pore pressures than coarse‐grained sediments, resulting in lower critical angles for slumping. To test the thaw slumping model quantitatively, I constructed a process‐based numerical model that includes thaw slumping, lacustrine sediment dispersal, and thaw‐driven lake floor subsidence. The model predicts that lake orientations and aspect ratios are controlled by topographic aspect and slope, not by wind direction and intensity. The thaw slumping model further predicts inverse correlations between lake area and bank height and between lake area and bank material texture. An analysis of oriented thaw lakes in northern Alaska shows that systematically smaller, deeper lakes form in coarse‐grained eolian sediments compared with those formed in fine‐grained fluvial marine sediments. This pattern strongly supports the thaw slumping model.
Comparison of two image reconstruction algorithms for microwave tomography
Authors: Fhager A. , Persson M.
Published: June 2005

Two image reconstruction algorithms for microwave tomography are compared and contrasted. One is a general, gradient‐based minimization algorithm. The other is the chirp pulse microwave computed tomography (CP‐MCT) method, which is a highly computationally efficient reconstruction method but also a method best suited for low contrasts. The results of the simulations show that when imaging high‐contrast objects, such as a breast cancer tumor, reconstructions made are comparable to results from the minimization algorithm below a contrast of about 10%. The simulations, however, show that the reconstructions made by the CP‐MCT method are very robust to noise. The reconstruction of the conductivity using the minimization algorithm, on the other hand, is very sensitive to the level of noise. In spite of a strong degradation in the conductivity reconstructions, the corresponding permittivity reconstructions do not show the same sensitivity to the noise level.
Temporal profile of T2-Weighted MRI Distinguishes between Pannecrosis and Selective Neuronal Death after Transient Focal Cerebral Ischemia in the Rat
Authors: Wegener Susanne , Weber Ralph , Ramos-Cabrer Pedro , Uhlenkueken Ulla , Sprenger Christiane , Wiedermann Dirk , Villringer Arno , Hoehn Mathias
Published: June 2005

Transient middle cerebral artery occlusion (MCAO) by an intraluminal thread leads to primarily subcortical infarctions with little sensorimotor impairment in the Wistar rat strain. We investigated the course of infarct development in this lesion type for 10 weeks using magnetic resonance imaging (MRI) along with histological characterization. MCAO was induced in male Wistar rats (260 to 300 g) for 60 mins. Animals received follow-up T1- and T2-weighted MRI from day 1 until week 10. Separate groups of animals were analyzed histologically after 2, 6, and 10 weeks. Histology included immunohistochemistry for neuronal and astrocytic markers as well as hematoxylin eosin and luxol fast blue-cresyl violet staining. In contrast to lesions involving the cortex, exclusively subcortical infarctions were characterized by a complete resolution of initially increased T1 and T2 relaxation times by 10 weeks. Between 2 and 10 weeks, neuronal death and gliosis as well as a dense inflammatory infiltrate were evident in these lesions, without damage to fiber tracts or development of cystic cavities. Exclusively subcortical lesions in Wistar rats are characterized by normalization of T1 and T2 relaxation times, which might, however, not be mistaken for tissue recovery. Despite this MRI normalization, selective neuronal death and gliosis develop. Although MRI at individual time points might therefore be ambiguous, the temporal profile of relaxation time changes over the chronic time period allows discrimination of the lesion development into selective neuronal death or pannecrosis.
Neurotoxic Zinc Translocation into Hippocampal Neurons is Inhibited by Hypothermia and is Aggravated by Hyperthermia after Traumatic Brain Injury in Rats
Authors: Suh Sang Won , Frederickson Christopher J , Danscher Gorm
Published: June 2005

Hypothermia reduces excitotoxic neuronal damage after seizures, cerebral ischemia and traumatic brain injury (TBI), while hyperthermia exacerbates damage from these insults. Presynaptic release of ionic zinc (Zn2+), translocation and accumulation of Zn2+ ions in postsynaptic neurons are important mechanisms of excitotoxic neuronal injury. We hypothesized that temperature-dependent modulation of excitotoxicity is mediated in part by temperature-dependent changes in the synaptic release and translocation of Zn2+. In the present studies, we used autometallographic (AMG) and fluorescent imaging of N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining to quantify the influence of temperature on translocation of Zn2+ into hippocampal neurons in adult rats after weight drop-induced TBI. The central finding was that TBI-induced Zn2+ translocation is strongly influenced by brain temperature. Vesicular Zn2+ release was detected by AMG staining 1 h after TBI. At 30°C, hippocampus showed almost no evidence of vesicular Zn2+ release from presynaptic terminals; at 36.5°C, the hippocampus showed around 20% to 30% presynaptic vesicular Zn2+ release; and at 39°C vesicular Zn2+ release was significantly greater (40% to 60%) than at 36.5°C. At 6 h after TBI, intracellular Zn2+ accumulation was detected by the TSQ staining method, which showed that Zn2+ translocation also paralleled the vesicular Zn2+ release. Neuronal injury, assessed by counting eosinophilic neurons, also paralleled the translocation of Zn2+, being minimal at 30°C and maximal at 39°C. We conclude that pathological Zn2+ translocation in brain after TBI is temperature-dependent and that hypothermic neuronal protection might be mediated in part by reduced Zn2+ translocation.
Intersubject Variability and Reproducibility of 15O PET Studies
Authors: Coles Jonathan P , Fryer Tim D , Bradley Peter G , Nortje Jurgens , Smielewski Peter , Rice Kenneth , Clark John C , Pickard John D , Menon David K
Published: June 2005

Oxygen-15 positron emission tomography (15O PET) can provide important data regarding patients with head injury. We provide reference data on intersubject variability and reproducibility of cerebral blood flow (CBF), cerebral blood volume (CBV), cerebral metabolism (CMRO2) and oxygen extraction fraction (OEF) in patients and healthy controls, and explored alternative ways of assessing reproducibility within the context of a single PET study. In addition, we used independent measurements of CBF and CMRO2 to investigate the effect of mathematical correlation on the relationship between flow and metabolism. In patients, intersubject coefficients of variation (CoV) for CBF, CMRO2 and OEF were larger than in controls (32.9% ± 2.2%, 23.2% ± 2.0% and 22.5% ± 3.4% versus 13.5% ± 1.4%, 12.8% ± 1.1% and 7.3% ± 1.2%), while CoV for CBV were lower (15.2% ± 2.1% versus 22.5%±2.8%) ( P>0.001). The CoV for the test—retest reproducibility of CBF, CBV, CMRO2 and OEF in patients were 2.1% ± 1.5%, 3.8% ± 3.0%, 3.7% ± 3.0% and 4.6% ± 3.5%, respectively. These were much lower than the intersubject CoV figures, and were similar to alternative measures of reproducibility obtained by fractionating data from a single study. The physiological relationship between flow and metabolism was preserved even when mathematically independent measures were used for analysis. These data provide a context for the design and interpretation of interventional PET studies. While ideally each centre should develop its own bank of such data, the figures provided will allow initial generic approximations of sample size for such studies.
Molecular dynamics investigation of the structural properties of phosphatidylethanolamine lipid bilayers
Authors: Suits Frank , Pitman Michael C. , Feller Scott E.
Published: June 2005

We report a 14 ns microcanonical (NVE) molecular dynamics simulation of a fully hydrated bilayer of 1-stearoyl-2-oleoyl-phosphatidyethanolamine. This study describes the structure of the bilayer in terms of NMR order parameters and radial distribution functions, and compares them to experimental results and simulations of other lipids. A focus of this work is the characterization of the lipid-water interface, particularly the hydrogen bonding network of the phosphatidylethanolamine (PE) headgroups. We find that hydrogen bonding between the primary amine and phosphate groups has a pronounced effect on the structure of PE relative to phosphatidylcholine, and is evident in, for example, the P–N radial distribution functions.
Molecular dynamics investigation of dynamical properties of phosphatidylethanolamine lipid bilayers
Authors: Pitman Michael C. , Suits Frank , Gawrisch Klaus , Feller Scott E.
Published: June 2005

We describe the dynamic behavior of a 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) bilayer from a 20ns molecular dynamics simulation. The dynamics of individual molecules are characterized in terms of H2 spin-lattice relaxation rates, nuclear overhauser enhancement spectroscopy (NOESY) cross-relaxation rates, and lateral diffusion coefficients. Additionally, we describe the dynamics of hydrogen bonding through an analysis of hydrogen bond lifetimes and the time evolution of clusters of hydrogen bonded lipids. The simulated trajectory is shown to be consistent with experimental measures of internal, intermolecular, and diffusive motion. Consistent with our analysis of SOPE structure in the companion paper, we see hydrogen bonding dominating the dynamics of the interface region. Comparison of H2 T1 relaxation rates for chain methylene segments in phosphatidylcholine and phosphatidylethanolamine bilayers indicates that slower motion resulting from hydrogen bonding extends at least three carbons into the hydrophobic core. NOESY cross-relaxation rates compare well with experimental values, indicating the observed hydrogen bonding dynamics are realistic. Calculated lateral diffusion rates (4±1×10−8cm2∕s) are comparable, though somewhat lower than, those determined by pulsed field gradient NMR methods.