bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations of chromosomal copy number during progression of diffuse‐type gastric carcinomas: metaphase‐ and array‐based comparative genomic hybridization analyses of multiple samples from individual tumours
Authors: Peng Dun‐Fa , Sugihara Hiroyuki , Mukaisho Ken‐ichi , Tsubosa Yasuhiro , Hattori Takanori
Published: October 2003

The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on gastric carcinoma (GC), but there is little information on the time sequence of cytogenetic changes. In the present study, degenerate oligonucleotide‐primed polymerase chain reaction (DOP‐PCR) and CGH were applied to multiple samples microdissected from 19 diffuse‐type GCs including eight early cancers. Recurrent gains were detected at 8q, 3q, 7q, and 8p, and loss at 17p (in more than 50% of the cancers), the frequencies of which were fairly similar between the samples with (SIG) and those without (POR) abundant signet ring cells. Earlier stemline changes (8q+, 8p+, 1q+, 17p−, etc), with breakpoints that were common to all the samples, were discriminated from later sideline changes (2q+, 11q+, 17q−, 21q−, etc) in individual tumours. The changes were generally common to early and advanced cancers, except for 7p+, 15q+, 3p−, and 18q−, which were largely sideline changes and more frequently detected in advanced cancers (p < 0.05). Because the samples with 7p+ had a greater number of copy‐number changes than those without 7p+ (p < 0.01), 7p+ may play a role in tumour progression by acceleration of chromosomal instability. Fifteen different chromosomal loci with amplification were detected in ten cases, mostly as sideline changes in advanced cancers. By microarray‐based CGH, KRAS, MDM2, and FGFR2 were confirmed in the amplicons at 12p, 12q, and 10q, and FES at 15q26, for the first time in GC. These results support the notion that SIG and POR are of a genetically single lineage in both early and advanced diffuse‐type GC and that the majority of advanced cancers derive from early cancers through the accumulation of various sideline changes in addition to stemline changes. Copyright © 2003 John Wiley & Sons, Ltd.
Is high‐grade adenomatous hyperplasia an early bronchioloalveolar adenocarcinoma?
Authors: Ullmann R , Bongiovanni M , Halbwedl I , Fraire AE , Cagle PT , Mori M , Papotti M , Popper Helmut H
Published: October 2003

Atypical adenomatous hyperplasia (AAH) is a probable forerunner of bronchioloalveolar carcinoma (BAC) and pulmonary adenocarcinoma (AC) of mixed type. The present study analysed four low‐grade AAHs, 13 high‐grade AAHs, two BACs, nine mixed ACs, and one squamous cell carcinoma derived from 13 patients using comparative genomic hybridization. The average number of chromosomal aberrations was 1.2 in low‐grade AAH, 9.6 in high‐grade AAH, and 12.5 in AC. A high degree of overlap of genetic changes was found in high‐grade AAH, BAC, and AC within individual patients. The high number of aberrations and the degree of shared aberrations found in high‐grade AAH and AC raises questions about the separation of these two entities. In addition, in view of the monoclonal origin of multiple foci within the same patient, AAH may not be a precursor of AC in some cases, but rather may represent intraepithelial spread. Copyright © 2003 John Wiley & Sons, Ltd.
Apoptotic gene expression in Paget's disease: a possible role for Bcl‐2
Authors: Brandwood Craig P , Hoyland Judith A , Hillarby M Chantal , Berry Jacqueline L , Davies Michael , Selby Peter L , Mee Andrew P
Published: October 2003

Paget's disease of bone is characterized by an increase in both the size and the number of bone‐resorbing osteoclasts. An important regulator of osteoclast activity is the process of apoptosis, and any aberration in this process could lead to increased osteoclasis. Analysis using human apoptosis cDNA expression arrays revealed that the apoptotic suppressor, Bcl‐2, showed a marked increase in expression in Pagetic bone. In situ hybridization (ISH) and computer‐assisted image analysis confirmed that the levels of Bcl‐2 transcripts were significantly (p < 0.0001) increased in Pagetic osteoclasts. The Bcl‐2 : Bax transcript ratios were similarly elevated. These findings were confirmed by immunohistochemistry. The Bcl‐2 gene promoter sequence from 20 Pagetic patients and controls was analysed. Single nucleotide mutations were identified in three of the Paget's patients and one of the controls. Luciferase reporter analysis showed that the mutations induced a basal 12‐fold increase and hydrogen peroxide‐induced 19‐fold increase in luciferase expression, compared with the normal construct. It is concluded that in Paget's disease, there is an increase in the expression of genes that are involved in the inhibition of apoptosis, notably Bcl‐2. The increase in Bcl‐2 may be explained in some patients by mutations in the Bcl‐2 gene promoter. These results provide a potential explanation for the dramatic increase in osteoclasis seen in patients with Paget's disease. Copyright © 2003 John Wiley & Sons, Ltd.
Expression of p27kip1 in breast cancer and its prognostic significance
Authors: Barnes A , Pinder SE , Bell JA , Paish EC , Wencyk PM , Robertson JFR , Elston CW , Ellis IO
Published: October 2003

p27kip1 is a member of the KIP/CIP family of cyclin‐dependent kinase inhibitors and is a negative cell‐cycle regulator that is thought to play a role in tumour suppression. Reduced levels of this protein have been observed in a number of human cancers. However, evidence is conflicting as to whether p27kip1 has a role to play in breast cancer, including predicting behaviour and prognosis. The present investigation aimed to provide a definitive study of 830 breast cancer cases with median patient follow‐up of 104 months to determine the true prognostic significance, if any. Immunohistochemical analysis of tissue microarrays and three scoring methods were used to assess p27kip1 expression. Univariate analysis showed a significant relationship between reduced p27kip1 expression and increasing tumour grade, nuclear pleomorphism, mitosis, and decreasing tubule formation (allp< 0.001). Significant associations between reduced p27, negative oestrogen receptor status, and ductal/no special type tumours were also observed. Survival analysis demonstrated that patients with tumours with high p27kip1 levels had an improved survival compared with those with cancers with low expression. On multivariate analysis, when compared with existing factors, p27kip1 was not, however, an independent prognostic factor. It is concluded that the inverse relationship between p27kip1 levels and histological grade and individual grade components suggests a role for p27kip1 in both cell proliferation and differentiation, but is not clinically useful. Copyright © 2003 John Wiley & Sons, Ltd.
Gastric cancer: the role of insulin‐like growth factor 2 (IGF 2) and its receptors (IGF 1R and M6‐P/IGF 2R)
Authors: Pavelić Krešimir , Kolak Toni , Kapitanović Sanja , Radošević Senka , Spaventi Šime , Krušlin Božo , Pavelić Jasminka
Published: October 2003

Insulin‐like growth factor 2 (IGF 2) appears to be involved in the progression of many tumours. It binds to at least two different types of receptor: IGF type 1 (IGF 1R) and mannose 6‐phosphate/IGF type 2 (M6‐P/IGF 2R). Ligand binding to IGF 1R provokes mitogenic and anti‐apoptotic effects. M6‐P/IGF 2R has a tumour suppressor function—it mediates IGF 2 degradation. Mutation of M6‐P/IGF 2R causes both diminished growth suppression and augmented growth stimulation. The aim of this study was to investigate the role of IGF 2 and its receptors (IGF 1R and IGF 2R) in human gastric cancer. The expression of IGF 2 and its receptors was measured in order to analyse the possible correlation between the activity of these genes and cell proliferation in two different gastric tumour types: diffuse and intestinal. The effect of IGF 1 receptor blockage on cell proliferation and anchorage‐independent cell growth was also examined. Increased expression of IGF 2 and IGF 1R genes (at the mRNA and protein level) was found in gastric cancer when compared with non‐tumour tissue. Furthermore, there was a significant difference between IGF 2 expression in the more aggressive diffuse type and that in the intestinal type of gastric cancer. Moreover, the IGF 2 peptide level in the culture media obtained from the diffuse type of cancer cells was significantly higher when compared with the intestinal type. The level of IGF 2 peptide in the conditioned media strongly correlated with [3H]thymidine incorporation and cell proliferation. On the contrary, IGF 2R mRNA expression was much higher in the intestinal type of cancer than in the diffuse type. In addition, IGF 2R protein expression was substantially lower with progression of the diffuse cancer type to a higher stage. The αIR3 monoclonal antibody strongly inhibited [3H]thymidine incorporation and decreased the number of colonies in soft agar of cells overexpressing IGF 2. These findings suggest that members of the IGF family are involved in the pathogenesis of gastric cancer, probably by autocrine/paracrine stimulation of cell growth. Such tumours might be excellent candidates for therapeutic strategies aimed at interference with this pathway. Copyright © 2003 John Wiley & Sons, Ltd.
Chromosomal imbalances: a hallmark of tumour relapse in primary cutaneous CD30+ T‐cell lymphoma
Authors: Prochazkova Martina , Chevret Edith , Beylot‐Barry Marie , Sobotka Jiri , Vergier Béatrice , Delaunay Michèle , Turmo Michèle , Ferrer Jacky , Kuglik Petr , Merlio Jean‐Philippe
Published: October 2003

Primary cutaneous CD30+ large T‐cell lymphoma (CD30+ CTCL) is a subset of non‐epidermotropic primary cutaneous T‐cell lymphoma. Although frequent spontaneous regression may be observed, skin relapses occur frequently. Cytogenetic abnormalities that could play a role in CD30+ CTCL tumour pathogenesis and relapses remain unknown. The identification of recurrent cytogenetic abnormalities is hampered by difficulty in culturing tumours and the lack of CD30+ CTCL serial studies comparing genetic changes both at diagnosis and at relapse. The purpose of this study was to investigate the cytogenetic abnormalities present in a series of 13 CD30+ CTCL samples obtained from nine patients fulfilling both EORTC and WHO diagnostic criteria, by the use of comparative genomic hybridization (CGH). CGH analysis revealed a non‐random distribution of genetic imbalances between relapsing and non‐relapsing disease. In relapsing disease, chromosomal abnormalities were detected both in the primary tumour and in relapses. The mean number of changes in non‐relapsing disease was 0.33 (range 0–1), compared with 6.29 (range 1–16) in relapsing disease. The recurrent chromosomes involved in relapsing disease were chromosomes 6 (86%), 9 (86%), and 18 (43%). While chromosome 9 was mostly affected by gain, chromosomes 6 and 18 mainly contained regions of loss, exclusively on 6q and 18p. The common regions of deletion were 6q21 and 18p11.3. In one patient, we successfully cultured tumour cells from a skin biopsy from a second relapse. The G‐banded karyotype was concordant with both CGH and fluorescence in situ hybridization (FISH) results. Although further studies are required to strengthen these data, this CGH analysis demonstrates chromosomal imbalances that may be involved in the pathogenesis of relapsing CD30+ CTCL. Copyright © 2003 John Wiley & Sons, Ltd.
Loss of parental‐specific methylation at the IGF2 locus in human hepatocellular carcinoma
Authors: Poirier Karine , Chalas Céline , Tissier Frédérique , Couvert Philippe , Mallet Vincent , Carrié Alain , Marchio Agnès , Sarli Dario , Gicquel Christine , Chaussade Stanislas , Beljord Cherif , Chelly Jamel , Kerjean Antoine , Terris Benoit
Published: October 2003

Significant production of the growth factor IGF2 has been reported in human hepatocellular carcinomas (HCCs). Disturbances associated with changes in methylation at this locus or affecting the 11p15.5 imprinting domain as a whole can be postulated in HCCs. In the present study, the methylation status of differentially methylated regions of the imprinted genes TSSC5, LIT1, and IGF2, which span the 11p15 domain, was analysed in 71 liver tissues from virus‐associated and non‐virus‐associated HCCs compared with six normal liver tissues. Altered methylation of TSSC5 and LIT1 was observed in only 6% and 8% of HCCs, respectively, compared with 89% at the IGF2 locus, suggesting that these loci were not concomitantly dysregulated. These observations suggest that loss of parental‐specific methylation at the IGF2 locus may be specifically associated with HCC, whether virus‐associated or non‐virus‐associated, and arising in cirrhotic or non‐cirrhotic livers. Copyright © 2003 John Wiley & Sons, Ltd.
Stellenwert der CSE‐Hemmer: Prophylaxe und Therapie der KHK
Authors: Kostner Karam
Published: October 2003

Man kann festhalten, dass es keinen Zweifel am klinischen Benefit einer LDL‐C‐Senkung mittels Statinen geben kann. Die im letzten Jahr veröffentlichten ATP‐III‐Richtlinien stellen eine gute und praxisnahe Hilfestellung für die Therapieentscheidung dar. Die Entscheidung ist aber immer individuell zu treffen und obliegt letztendlich der klinischen Beurteilung des behandelnden Arztes. Die wichtige Frage, ob eine noch aggressivere LDL‐C‐Senkung zu niedrigeren Zielwerten (unter 80 mg/dL) einen zusätzlichen Nutzen bringt, ist noch nicht beantwortet. Wenn ein solcher Zusatznutzen besteht, so muss er jedenfalls gegen das möglicherweise erhöhte Nebenwirkungs‐Risiko einer besonders aggressiven, hochdosierten Statintherapie abgewogen werden. Es ist zu erwarten, dass die Ergebnisse laufender randomisierter Doppelblind‐Studien zur Klärung dieser Frage wesentlich beitragen werden. Die Ergebnisse der HPS‐Studie legen den Schluss nahe, dass bei Hochrisikopatienten eine LDL‐Senkung im Ausmaß von 30 bis 40 % in jedem Fall erfolgen sollte, auch wenn ein niedriger Ausgangswert des LDL‐C vorliegt. Zusammenfassend kann gesagt werden, dass Statine eine sehr wichtige Rolle in der Primär‐ und Sekundärprävention der Koronaren Herzkrankheit spielen und daher gewissermaßen das Penicillin der Atherosklerosetherapie darstellen.
Riboflavin als Sonnenschutz
Authors: Gießen Hannelore
Published: October 2003

Vitamine werden in großer Menge gebraucht. Nicht nur in der Humanmedizin für Arzneimittel, Vitaminsupplemente und Nahrungsergänzungsmittel, sondern auch für die Futtermittelindustrie. Eines der B‐Vitamine, das Riboflavin, wird inzwischen auf einem interessanten Weg von Mikroorganismen hergestellt.
Water sorption into poly[(2‐hydroxyethyl methacrylate)‐co‐(1‐vinyl‐2‐pyrrolidone)]at 310 K
Authors: Malak Mark , Hill David JT , Whittaker Andrew K
Published: October 2003

Poly(2‐hydroxyethyl methacrylate) and copolymers of 2‐hydroxyethyl methacrylate (HEMA) and 1‐vinyl‐2‐pyrrolidone (VP) in the form of cylindrical samples (≈8 mm × 20 mm) have been prepared and the sorption of water into these cylinders has been studied by the mass‐uptake methods and by magnetic‐resonance imaging. The equilibrium water contents for the cylinders were found to vary systematically with the copolymer composition. Diffusion of water into the cylinders was found to follow Fickian behaviour for cylinders with high HEMA contents, with the diffusion coefficients obtained from mass‐uptake studies dependent on the copolymer composition, varying from 1.7 × 10−11 m2 s−1 for poly(HEMA) to 2.0 × 10−11 m2 s−1 for poly(HEMA‐co‐VP) with a composition of 1:1. However, NMR‐imaging studies showed that, while the profiles of the water diffusion fronts for cylinders with high HEMA contents were Fickian, that for the 1:1 copolymer was not and indicated that the mechanism was Case III. The polymers which were rich in VP were characterized by a water‐sorption process which follows Case‐III behaviour. Copyright © 2003 Society of Chemical Industry
Calorimetric and rheological characterization of a high‐performance epoxy curable at low temperatures
Authors: Rocks J , Halter M , George G , Vohwinkel F
Published: October 2003

Curing and gelation ofN,N,N′,N′‐tetraglycidyl‐4,4′‐diaminodiphenylmethane (TGDDM, Araldite®MY721) in the presence of an anhydride hardener mixture were studied by calorimetric and dynamic rheological measurements. The cure kinetics of the homogeneous and low‐temperature‐curable epoxy system were evaluated from 40 to 140 °C, and the apparent activation energies of the curing reaction were calculated from different thermokinetic models. By comparison of different calorimetric and rheological methods, it was found that the results from the isothermal calorimetric measurements are in good agreement with the results obtained from isothermal rheological experiments, while the kinetic parameter calculations based on dynamic calorimetric measurements are not reliable results. These differences mainly arise from the fact that dynamic measurements have many underlying assumptions and do not consider the equilibrium condition. Copyright © 2003 Society of Chemical Industry
Curing kinetics and thermomechanical behaviour of co‐anhydride cured aminoglycidyl epoxy resins
Authors: Rocks Jens , George Graeme A , Vohwinkel Friedrich
Published: October 2003

The curing behaviour and thermomechanical properties of a technical grade ofN,N,N′,N′‐tetraglycidyl‐4,4′‐diaminodiphenylmethane (TGDDM, Araldite®MY721), cured in the presence of an anhydride hardener mixture consisting of maleic anhydride (MA) and pyromellitic acid dianhydride (PMDA) was studied by calorimetric and dynamic mechanical analysis. The cure kinetics and the influence of varying stoichiometric anhydride‐to‐epoxy ratios were evaluated and the apparent activation energy was calculated according to Barrett's method. High extents of conversion from DSC studies of the MY721‐resin were reached after room temperature cure, without an added catalyst, for 24 h, followed by a post‐curing step of 1 h at 90 °C. The ultimate glass transition temperatures for the molar anhydride/epoxy ratio,r= 0.8, were close to the decomposition temperature, and indications were obtained that the network structure consists of two independent sub‐networks. It is suggested that two separate mechanisms contribute to the curing reaction at room temperature. First, the tertiary amine structure, intrinsic to aminoglycidyl resins, may act as an internal catalyst for the anhydride ring opening, and secondly, the unsaturated bond of MA participates in the curing reaction by nucleophilic attack, such as from tertiary amines or carboxylate or alkoxide anions. From a study of a range of different aminoglycidyl resins, this low‐temperature curing behaviour is found to be a general phenomenon. Copyright © 2003 Society of Chemical Industry
Yield behaviour of a melt‐compounded polyethylene–intercalated montmorillonite nanocomposite
Authors: Truss Rowan W , Lee Ang Chiean
Published: October 2003

The yield behaviour of a series of melt‐mixed polyethylene–modified montmorillonite nanocomposites has been studied as a function of temperature and strain rate and compared to the behaviour of the base polymer. The processing conditions used gave an intercalated structure as assessed by X‐ray diffraction. Although there was a modest improvement in stiffness with clay content, the yield behaviour was insensitive to the addition of the clay. Both the base polymer and the nanocomposites showed double yield points. These were analysed as activated rate processes, with the activation energies consistent with the low strain yield point being associated with the α2 molecular relaxation and the higher strain yield point with ‘c’ axis slip. Copyright © 2003 Society of Chemical Industry
Initiation in the free radical (co)polymerization of dialkyl fumarates and dicyclohexyl fumarate/tert‐butyl vinyl ether investigated by a nitroxide trapping technique
Authors: Hirano Emari , Zetterlund Per B , Takaoka Toshiaki , Yamada Bunichiro , Buckley Robbie , Jenkins Ian D , Busfield W Ken
Published: October 2003

Initiation in the free radical polymerization of dicyclohexyl fumarate (DCHF), isopropyl phenyl fumarate (iPPF) and isopropyl benzyl fumarate (iPBF), and the copolymerization of DCHF with tert‐butyl vinyl ether (E), initiated by di‐tert‐butyl peroxalate, has been investigated at 60 °C. The rate coefficient for addition of the tert‐butoxy radical to the fumarates is of the order 105 M−1 s−1 with differences between the different fumarates being at most a factor of about two. A significant amount of monomer addition to the primary propagating radical in the case of DCHF occurred despite competition with trapping, indicating that the first propagation step is orders of magnitude faster than the propagation step for macroradicals.

In the copolymerization system DCHF/E, second‐generation carbon‐centered radicals containing two fumarate units were formed, whereas the corresponding species in the homopolymerization of E was not observed. This is consistent with the fact that E does not undergo homopolymerization. Copyright © 2003 Society of Chemical Industry
Reaction behavior of sterically hindered α‐(substituted methyl)acrylic esters with tert‐butoxy radicals studied by a nitroxide trapping technique
Authors: Sato Eriko , Zetterlund Per B , Yamada Bunichiro , Busfield W Ken , Jenkins Ian D
Published: October 2003

The nitroxide free radical trapping technique in connection with HPLC and electrospray ionization mass spectrometry has been employed to study reactions of tert‐butoxy radicals with methyl α‐(2‐methyl‐2‐carbomethoxypropyl)acrylate (methyl methacrylate dimer (MMAD)) and methyl α‐(2,2,2‐tris(carbomethoxy)ethyl)acrylate (M(TM)EA). It has been shown that the adduct radical derived from MMAD undergoes β‐fragmentation and expels the 2‐carbomethoxy‐2‐propyl radical. No fragmentation was detected in the case of M(TM)EA. Rate coefficients of trapping and β‐fragmentation of the tert‐butoxy radical in the literature have enabled estimations of absolute rate coefficients for additions of the tert‐butoxy radical and the methyl radical to monomer, hydrogen abstraction from monomer by the tert‐butoxy radical, and β‐fragmentation of the adduct radical (MMAD only). The reactions of tert‐butoxy radicals with M(TM)EA are slower than with MMAD due to the steric hindrance of the α‐substituent. Copyright © 2003 Society of Chemical Industry
A kinetic study of the thermally initiated free radical reactions of styrene with octanedithiol as a model for the reactions of multifunctional thiols with divinylbenzene
Authors: Ang Julie B , Hill David JT , Pomery Peter J , Toh Huan
Published: October 2003

The kinetics of chain reactions of octanedithiol with styrene, thermally initiated with TX29B50 (a 50:50 wt% solution of TX29 diperoxy initiator in a phthalate plasticizer), have been studied over a range of initiator concentrations, a range of mixture formulations and a range of temperatures. This system has been investigated as a model system for the reactions of polyfunctional thiols with divinyl benzene. The reactions have been shown to follow first‐order kinetics for both the thiol and the ene species and to be characterized by a dependence on the initiator concentration to the power of one half. The kinetic rate parameters have been shown to adhere to Arrhenius behaviour. A kinetic model for the chain reactions for this system has been proposed. Copyright © 2003 Society of Chemical Industry
Simultaneous FT‐NIR and ESR analyses to yield propagation rate coefficients for polymerization of methyl methacrylate based monomers
Authors: Le Tri T , Hill David JT
Published: October 2003

A hyphenated instrumental approach has been used to obtain reliable values for the propagation rate coefficients as a function of conversion for polymerizations of methyl methacrylate (MMA) and a mixture of MMA and ethyleneglycol dimethacrylate (EGDMA) with a 1:1 concentration of double bonds, from near the onset of the Trommsdorf region into the glass region. ESR spectroscopy was used to measure the radical concentration while FT‐NIR fibre‐optic spectroscopy was employed to measure instantaneously the double‐bond concentration within the temperature‐controlled cavity of the ESR instrument during polymerization. The advantage of this approach to the measurement of the rate coefficient is that it is equally applicable to branching and linear polymerizations. For the polymerization of methyl methacrylate, the values of the rate coefficient at the lowest conversions at which reliable values could be obtained were in agreement with recently reported values obtained by the PLP‐SEC method. For the lowest conversions, the values obtained were 403 l mol−1 s−1 at 306 K for MMA and 520 l mol−1 s−1 at 310 K for a 1:1 mixture of MMA and EGDMA. Copyright © 2003 Society of Chemical Industry
A study of the radiation degradation of styrene/alkane and α‐methylstyrene/alkane copolymers
Authors: Cardona Francisco , Hill David JT , Pomery Peter J , Whittaker Andrew K
Published: October 2003

The effects of copolymer composition and microstructure on the radiation chemistry of styrene/alkane and α‐methylstyrene/alkane copolymers have been studied. The primary radical species formed on radiolysis of the copolymers at 77 K, and identified by ESR spectroscopy, are the same as those formed during radiolysis of the homopolymers. The yields of radicals for the copolymer are as predicted assuming that the cross‐section is proportional to the electron density of each component; however, there is some evidence of radical migration to aromatic groups at 77 K. Changes in molecular structure on irradiation were detected by using 13C NMR spectroscopy. Evidence of the consumption of terminal double bonds, and chain scission in α‐methylstyrene/alkane copolymers was found. Measurements of viscosity supported the mechanism of cross‐linking predominating in styrene/alkane copolymers, while in α‐methylstyrene/alkane copolymers chain scission was the major result of irradiation. Copyright © 2003 Society of Chemical Industry
Characterisation of grafted supports used for solid‐phase synthesis
Authors: Blakey Idriss , Day Gary , Girjes Ebtihal , Hunter David S , Rasoul Firas
Published: October 2003

A series of ‘pellicular’ type supports were fabricated by direct gamma‐radiation‐mediated graft polymerisation of styrene onto polypropylene, followed by aminomethylation. Raman spectroscopy was used for measuring the level of penetration of polystyrene graft into polypropylene, and other structural features such as density of graft and depth of functionalisation. The kinetics of the coupling of fluorenylmethylcarbamate (Fmoc)‐labelled amino acids, to the aminomethylated polystyrene grafts have been measured by UV absorption followed cleavage of the Fmoc chromophore. The Raman spectroscopy results showed that for this series of experiments the calculated rate coefficient for coupling of Fmoc‐labelled amino acids was primarily dependent on graft thickness, but was also influenced by the proportion of polystyrene graft to polypropylene. In general, it was also shown that with increasing loading capacity of support the calculated rate coefficient for amino‐acid coupling decreased correspondingly. In addition, a support that had both a high rate coefficient and a high loading capacity was prepared from polypropylene base material with a co‐continuous porous structure (high surface area). Copyright © 2003 Society of Chemical Industry
Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma
Authors: Yu Han‐Ling , Chertkow Howard M. , Bergman Howard , Schipper Hyman M.
Published: October 2003

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin‐agarose (HepA) and concanavalin A‐agarose (ConA) columns followed by separation on one‐dimensional and two‐dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in‐strip derivatization with 2,4‐dinitrophenylhydrazine (DNP) and anti‐DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA‐ConA 2‐D gels relative to NEC samples were analyzed by matrix‐assisted laser desorption‐time of flight‐mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and α‐1‐antitrypsin (α‐1‐AT). These glycoproteins were concentrated, respectively, 5‐, 6.5‐ and 107‐fold in HepA‐ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and α‐1‐AT as potential biological markers of this condition.
Proteomics of the dissimilatory iron‐reducing bacterium Shewanella oneidensis MR‐1, using a matrix‐assisted laser desorption/ionization‐tandem‐time of flight mass spectrometer
Authors: Vanrobaeys Frank , Devreese Bart , Lecocq Elke , Rychlewski Leszek , De Smet Lina , Van Beeumen Jozef
Published: October 2003

Shewanella oneidensis MR‐1 is a gram‐negative facultative aerobic bacterium living at oxic‐anoxic interfaces in nature. The plasticity of terminal electron‐acceptors used under anaerobic conditions is huge, but the adaptation to these different environmental conditions remains unclear. In this work, we used a proteomic approach to study the protein content when the organism is grown under anaerobic respiration conditions on insoluble ferric oxide. By analysis of two‐dimensional gel patterns of soluble protein extracts, we discovered 20 differentially displayed proteins. The protein spots were further analyzed by mass spectrometry for which we used, in addition to nano‐high‐performance liquid chromatography coupled to an electrospray ionization‐quadrupole‐time of flight instrument, a recently introduced matrix‐assisted laser desorption/ionization (MALDI) tandem‐time of flight mass spectrometer. The instrument allows the acquisition of high quality spectra, in both the mass spectrometry and tandem mass spectrometry mode, and is therefore able to identify protein spots unambiguously. Advantageous to electrospray ionization is a minimised sample handling, inherent to MALDI ionization, and the presence of high energy fragmentation ions, generating sequence information that also can differentiate isobaric amino acids. With this strategy, we could point out a regulatory protein that is up‐regulated under iron(III) respiration. This protein, the aerobic respiration control protein (ArcA), has been reported as being a regulator during anaerobiosis in other species. To our knowledge, this is the first report of the possible involvement of ArcA from S. oneidensis MR‐1 in the reduction of ferric oxide.
Proteome analysis of the rat cornea during angiogenesis
Authors: Thompson Larry J. , Wang Feng , Proia Alan D. , Peters Kevin G. , Jarrold Brad , Greis Kenneth D.
Published: October 2003

Angiogenesis is the formation of new blood vessels from the pre‐existing vasculature. However, the study of this complex process is often hampered by the lack of a suitable cell‐based model and the tools to study the biochemical events that lead to new blood vessel growth. The most widely accepted model for angiogenesis is the in vivo rat corneal model. In this model, the cornea, which is normally an avascular tissue, is stimulated to undergo angiogenesis in response to silver nitrate cauterization or to the implantation of an exogenous angiogenic agent. The physical changes associated with the new vessel growth can be readily monitored visually, but the regulated biochemical events that result in the growth and remodeling of the new vessels are much more challenging to decipher. In this report, a proteomics approach is evaluated for its utility in deciphering the biochemical events during a time course of angiogenic stimulation. At various time points post‐silver nitrate cautery, corneas were harvested, solubilized, and analyzed by two‐dimensional gel electrophoresis. Protein expression profiles at the various stages of angiogenesis were compared to those of control corneas. One hundred and eleven differentially‐expressed proteins were identified by either matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry or liquid chromatography‐coupled electrospray ionization tandem mass spectrometry. Many of the proteins up‐regulated during the angiogenesis process were identified as blood‐related proteins, thus validating the development of functional blood vessels in the normally avascular tissue of the cornea. Furthermore, detection of differentially‐regulated proteins in cauterized versus control tissue clearly validated the utility of a proteomics approach to study this model of angiogenesis. However, in order to get at the key regulatory proteins in the angiogenesis process, it is clear that additional scale‐up and enrichment approaches will be required.
Approaches for the quantification of protein concentration ratios
Authors: Moritz Bernd , Meyer Helmut E.
Published: October 2003

The function of a protein is modulated by its abundance and its degree of specific post‐translational modifications such as phosphorylation, glycosylation or truncation. Consequently, changes of protein concentration and the extent of their post‐translational modifications has a great influence on the activity of intracellular substrate degradation processes, on the activity of intracellular biosynthetic pathways, on the cell cycle or on the function of a single cell in a whole organism. Defects in this area lead to diseases like cancer or neurodegeneration. Therefore, it is a challenge to quantify changes within the proteome in the diseased state or between developmental stages and to use the results obtained for the maximization of product yields in biotechnology or for the development of new drug targets to fight against diseases. In order to determine the intracellular concentration of a protein it is necessary to spike the cell sample with the same protein in a pure form. If the concentration changes of many proteins have to be determined, it takes a long time to obtain all these proteins in a pure form. Therefore, most approaches in this field are restricted to the determination of protein abundance ratios between two states such as diseased or healthy tissues. In this case the proteins in the sample of state A function as an internal standard for the proteins in the sample of state B and vice versa. The most common techniques in this field are the comparison of two‐dimensional gel spot intensities after staining or the integration of mass spectrometric peak intensities after stable isotope labelling with 13C, 15N, 18O or deuterium. The results, advantages and drawbacks of these approaches are discussed. Stable isotope labelling in combination with mass spectrometry is more accurate than the comparison of spot intensities and has the potential for the investigation of highly complex tissue samples.
Signal pathway profiling of ovarian cancer from human tissue specimens using reverse‐phase protein microarrays
Authors: Wulfkuhle Julia D. , Aquino Joy A. , Calvert Valerie S. , Fishman David A. , Coukos George , Liotta Lance A. , Petricoin Emanuel F.
Published: October 2003

Defects in cell signaling pathways play a central role in cancer cell growth, survival, invasion and metastasis. An important goal of proteomics is to characterize and develop “circuit maps” of these signaling pathways in normal and diseased cells. We have used reverse‐phase protein array technology coupled with laser capture microdissection and phospho‐specific antibodies to examine the activation status of several key molecular “gates” involved in cell survival and proliferation signaling in human ovarian tumor tissue. The levels of activated extracellular‐regulated kinase (ERK1/2) varied considerably in tumors of the same histotype, but no significant differences between histotypes were observed. Advanced stage tumors had slightly higher levels of phosphorylated ERK1/2 compared to early stage tumors. The activation status of Akt and glycogen synthase kinase 3β, key proteins and indicators of the state of the phosphatidylinositol 3‐kinase/Akt pro‐survival pathway also showed more variation within each histotype than between the histoypes studied. Our results demonstrate the utility of reverse phase protein microarrays for the multiplexed analysis of signal transduction from discreet cell populations of cells procured directly from human ovarian tumor specimens and suggest that patterns in signal pathway activation in ovarian tumors may be patient‐specific rather than type or stage specific.
Identification of chromatin‐related protein interactions using protein microarrays
Authors: Coleman Matthew A. , Miller Kristi A. , Beernink Peter T. , Yoshikawa Daniel M. , Albala Joanna S.
Published: October 2003

Dynamic structural changes in chromatin are mediated by protein interactions that modulate multiple cellular processes including replication, transcription, recombination and DNA repair. Complexes that recognize chromatin are defined by several distinct groups of proteins that either directly modify histones or interact with histone‐DNA complexes. A protein microarray format was used to analyze the interaction of various DNA repair proteins with chromatin components. We applied proteins, antibodies and DNA to functionalized glass slides and interrogated the slides with our proteins of interest to identify novel protein‐protein interactions for proteins involved in DNA double‐strand break repair. Here we demonstrate that the DNA repair protein RAD51B, and not its cognate partner RAD51C, interacts with histones and not nucleosomes. Nucleosome‐specific interactions were demonstrated with the recently identified SWI/SNF protein, SMARCAL1. Unique RAD51B‐histone interactions were corroborated using Far Western analysis. This is the first demonstration of an interaction between RAD51B and histone proteins that may be important for the successful repair of DNA double‐strand breaks.
Molecular profiling of the immune response in colon cancer using protein microarrays: Occurrence of autoantibodies to ubiquitin C‐terminal hydrolase L3
Authors: Nam Myeong J. , Madoz‐Gurpide Juan , Wang Hong , Lescure Pascal , Schmalbach Cecelia E. , Zhao Rong , Misek David E. , Kuick Rork , Brenner Dean E. , Hanash Samir M.
Published: October 2003

We implemented a protein microarray approach to identify proteins that induce a humoral response in colon cancer. Solubilized proteins from the LoVo colon adenocarcinoma cell line were separated into 1760 fractions, arrayed onto nitrocellulose‐coated slides, and hybridized with individual sera from 15 newly diagnosed patients with colon cancer, 15 with lung cancer, and 15 healthy subjects. 39/1760 fractions showed enhanced reactivity with sera from patients with colon cancer (p < 0.01) relative to healthy controls. A distinct pattern of reactivity was observed with sera from colon cancer relative to lung cancer. One fraction that exhibited reactivity with 9/15 colon cancer sera was subjected to mass spectrometry leading to the identification of ubiquitin C‐terminal hydrolase isozyme 3 (UCH‐L3) as a constituent. To validate the occurrence of autoantibodies to UCH‐L3, independent analysis was done by means of Western blots. UCH‐L3 antibodies were detected in 19/43 sera from patients with colon cancer, and in 0/54 sera from subjects with lung cancer (24), colon adenoma (15) or otherwise healthy (15). Our findings indicate the occurrence of an immune response to a broad set of antigens in colon cancer and the feasibility of identifying the antigenic targets using a combination of protein microarrays and mass spectrometry.
Methods and applications of antibody microarrays in cancer research
Authors: Haab Brian B.
Published: October 2003

Antibody microarrays have great potential for significant value in biological research. Cancer research in particular could benefit from the unique experimental capabilities of this technology. This article examines the current state of antibody microarray technological developments and assay formats, along with a review of the demonstrated applications to cancer research. Work is ongoing in the refinement of various aspects of the protocols and the development of robust methods for routine use. Antibody microarray experimental formats can be broadly categorized into two classes: (1) direct labeling experiments, and (2) dual antibody sandwich assays. In the direct labeling method, the covalent labeling of all proteins in a complex mixture provides a means for detecting bound proteins after incubation on an antibody microarray. If proteins are labeled with a tag, such as biotin, the signal from bound proteins can be amplified. In the sandwich assay, proteins captured on an antibody microarray are detected by a cocktail of detection antibodies, each antibody matched to one of the spotted antibodies. Each format has distinct advantages and disadvantages. Several applications of antibody arrays to cancer research have been reported, including the analysis of proteins in blood serum, resected frozen tumors, cell lines, and on membranes of blood cells. These demonstrations clearly show the utility of antibody microarrays for cancer research and signal the imminent expansion of this platform to many areas of biological research.
Generation of bioreagents for protein chips
Authors: Phelan Michael L. , Nock Steffen
Published: October 2003

Protein microarrays have the potential to dramatically increase the throughput of proteomic analysis. Protein expression profiling chips with distinct spots of immobilized protein capture agents will allow the simultaneous measurement of hundreds to thousands of proteins from one sample. In contrast to DNA chips, for which the capture probes are easily designed and synthesized, the development of content for protein biochips is a long and laborious process. Careful consideration must be given to the specificities desired, the format of the assay, and the requirements of the capture agents, as well as to process optimization to minimize development time and cost. Monoclonal antibodies have been the prime choice as protein capture agents for the majority of protein chips developed to date. New technologies for the production of protein capture agents are more amenable to automation than traditional monoclonal antibody production and therefore carry the promise for industrialization.
Light directed massively parallel on‐chip synthesis of peptide arrays with t‐Boc chemistry
Authors: Gao Xiaolian , Zhou Xiaochuan , Gulari Erdogan
Published: October 2003

Peptide and peptidomimetic molecule arrays are emerging powerful tools for parallel screening of binding in proteomics and pharmaceutical discovery research. Up to now the common method of preparing peptide arrays was based on spotting on glass using a library of presynthesized peptides. However, due to the large number of monomers (amino acids) it is not possible to have combinatorial libraries which include all combinations of natural and synthetic amino acids. We describe a very flexible on‐chip oligopeptide synthesis method which uses the well developed t‐Boc based solid state synthesis chemistry. A very high degree of flexibility is achieved by using light photo generated acids and maskless projection lithography for spatially directed deprotection. Use of microfluidic chips enables moderately high densities, short reaction times and off‐the‐shelf chemicals. Examples are given from synthesis of metal ion binding peptides and epitope binding assys.
Microarrays to characterize protein interactions on a whole‐proteome scale
Authors: Schweitzer Barry , Predki Paul , Snyder Michael
Published: October 2003

Protein microarrays contain a defined set of proteins spotted and analyzed at high density, and can be generally classified into two categories; protein profiling arrays and functional protein arrays. Functional protein arrays can be made up of any type of protein, and therefore have a diverse set of useful applications. Advantages of these arrays include low reagent consumption, rapid interpretation of results, and the ability to easily control experimental conditions. The ultimate form of a functional protein array consists of all of the proteins encoded by the genome of an organism; such an array would be the whole proteome equivalent of the whole genome DNA arrays that are now available. While proteome microarrays may not have reached the stage of maturity of DNA microarrays, recent developments have shown that many of the barriers holding back the technology can be overcome. Arrays of this type have already been used to rapidly screen large numbers of proteins simultaneously for biochemical activities, protein‐protein interactions, protein‐lipid interactions, protein‐nucleic acid interactions, and protein‐small molecule interactions. Eventually, functional protein arrays will be used to facilitate various steps in the drug discovery and early development processes that are currently bottlenecks in the drug development continuum.
Effect of acidification treatment and morphological stability of sulfonated poly(arylene ether sulfone) copolymer proton‐exchange membranes for fuel‐cell use above 100 °C
Authors: Kim Yu Seung , Wang Feng , Hickner Michael , Mccartney Stephan , Hong Young Taik , Harrison William , Zawodzinski Thomas A. , Mcgrath James E.
Published: October 2003

Directly copolymerized wholly aromatic sulfonated poly(arylene ether sulfone) copolymers derived from 4,4′‐biphenol, 4,4′‐dichlorodiphenyl sulfone, 3,3′‐disulfonated, and 4,4′‐dichlorodiphenyl sulfone (BPSH) were evaluated as proton‐exchange membranes for elevated temperature operation (100–140 °C). Acidification of the copolymer from the sulfonated form after the nucleophilic step (condensation) copolymerization involved either immersing the solvent‐cast membrane in sulfuric acid at 30 °C for 24 h and washing with water at 30 °C for 24 h (method 1) or immersion in sulfuric acid at 100 °C for 2 h followed by similar water treatment at 100 °C for 2 h (method 2). The fully hydrated BPSH membranes treated by method 2 exhibited higher proton conductivity, greater water absorption, and less temperature dependence on proton conductivity as compared with the membranes acidified at 30 °C. In contrast, the conductivity and water absorption of a control perfluorosulfonic acid copolymer (Nafion 1135) were invariant with treatment temperature; however, the conductivity of the Nafion membranes at elevated temperature was strongly dependent on heating rate or temperature. Tapping‐mode atomic force microscope results demonstrated that all of the membranes exposed to high‐temperature conditions underwent an irreversible change of the ionic domain microstructure, the extent of which depended on the concentration of sulfonic acid sites in the BPSH system. The effect of aging membranes based on BPSH and Nafion at elevated temperature on proton conductivity is also discussed. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2816–2828, 2003
Thickness decrease of a grafted polyelectrolyte membrane exposed to shear flow
Authors: Kaneko D. , Narita T. , Gong J. P. , Osada Y. , Ando J. , Yamamoto K. , Ohnishi S. , Yaminsky V. V.
Published: October 2003

The effect of the shear flow on the thickness change of a polyelectrolyte membrane grafted onto a glass substrate was directly investigated with a flow cell combined with a confocal laser scanning microscope. The membrane thickness decreased proportionally to an increase in the shear stress of the flow when the shear rate exceeded a critical value of 1 s−1. The higher the ionic strength was of the fluid, the greater the thinning effect was. The correlation between the critical shear rate and the relaxation of the polymer in the gel membrane was examined. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2808–2815, 2003