bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous characterization of progenitor cell compartments in adult human liver
Authors: Porretti Laura , Cattaneo Alessandra , Colombo Federico , Lopa Raffaella , Rossi Giorgio , Mazzaferro Vincenzo , Battiston Carlo , Svegliati‐Baroni Gianluca , Bertolini Francesco , Rebulla Paolo , Prati Daniele
Published: December 2009

The human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic, and mesenchymal elements that probably derive from multiple lineage‐committed progenitors, but no comprehensive study aimed at identifying and characterizing intrahepatic precursors has yet been published. Cell suspensions for this study were obtained by enzymatic digestion of liver specimens taken from 20 patients with chronic liver disease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified, and characterized ex vivo according to previously validated methods, and then optimized flow cytometry was used to assess their relative frequencies and characterize their immunophenotypes in the clinical specimens. Stem and progenitor cells committed to hematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifiable in livers from both healthy and diseased subjects. Within the mononuclear liver cell compartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)+/CD49f+/CD29+/CD45−] accounted for 2.7–3.5% whereas hematopoietic (CD34+/CD45+), endothelial [vascular endothelial growth factor‐2 (KDR)+/CD146+/CD45−], and mesenchymal [CD73+/CD105+/CD90 (Thy‐1)+/CD45 −] stem cells and progenitors accounted for smaller fractions (0.02–0.6%). The patients' livers had higher percentages of hematopoietic and endothelial precursors than those of the donors. In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease. © 2009 International Society for Advancement of Cytometry
Steroid metabolism in chimeric mice with humanized liver
Authors: Lootens Leen , Van Eenoo Peter , Meuleman Philip , Pozo Oscar J. , Van Renterghem Pieter , Leroux‐Roels Geert , Delbeke Frans T.
Published: December 2009

Anabolic androgenic steroids are considered to be doping agents and are prohibited in sports. Their metabolism needs to be elucidated to allow for urinary detection by gas chromatography‐mass spectrometry (GC‐MS) or liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Steroid metabolism was assessed using uPA+/+ SCID mice with humanized livers (chimeric mice). This study presents the results of 19‐norandrost‐4‐ene‐3,17‐dione (19‐norAD) administration to these in vivo mice. As in humans, 19‐norandrosterone and 19‐noretiocholanolone are the major detectable metabolites of 19‐norAD in the urine of chimeric mice.

A summary is given of the metabolic pathways found in chimeric mice after administration of three model steroid compounds (methandienone, androst‐4‐ene‐3,17‐dione and 19‐norandrost‐4‐ene‐3,17‐dione). From these studies we can conclude that all major metabolic pathways for anabolic steroids in humans are present in the chimeric mouse. It is hoped that, in future, this promising chimeric mouse model might assist the discovery of new and possible longer detectable metabolites of (designer) steroids. Copyright © 2009 John Wiley & Sons, Ltd.
Spectrophotometric determination of peptic ulcer sulfur‐containing drugs in bulk form and in tablets
Authors: Mohamed Gehad G. , Nour El‐Dien F. A. , Khalil Shaban M. , El‐Tantawy Aliaa S. M.
Published: December 2009

Two spectrophotometric procedures are suggested for the determination of three irreversible proton pump inhibitors, rabeprazole (RAB), omeprazole (OMP) and pantoprazole (PAN) in pure form and in different pharmaceutical formulations. The first method is based on the oxidation of RAB and PAN with potassium iodate in an acidic medium followed by extracting the liberated iodine with cyclohexane and measurement at λ = 520 nm. Beer's law is valid in the concentration ranges from 10–400 and 5–400 µg ml−1 for RAB and PAN, respectively. The apparent molar absorptivities of the resulting coloured product were found to be 1.34 × 103 and 1.64 × 103 l.mol−1. cm−1 for RAB and PAN, respectively. The second method is based on the interaction of the basic drugs, OMP, RAB and PAN, in 1,2‐dichloroethane with bromophenol blue (BPB), bromocresol green (BCG) and bromocresol purple (BCP) in the same solvent to produce stable coloured ion pairs with maximum absorbance at 385–405 nm. Regression analysis of Beer's plots showed good correlation in the concentration ranges 10–60, 10–60 and 5–40 µg ml−1 for OMP, 10–150, 10–150 and 10–60 µg ml−1 for RAB and 10–250, 10–150 and 10–100 µg ml−1 for PAN with BPB, BCG and BCP reagents, respectively. The limits of detection are 0.46–7.69 µg ml−1 and limits of quantitation range between 1.52–8.53 µg ml−1. The optimum assay conditions were investigated and the recovery of the drugs from their dosage forms ranged from 99.33% to 100.5%. Intraday relative standard deviations (RSD) were 0.029–1.397% and the correlation coefficients ranged from 0.9992 to 1. The two methods can be applied successfully for the determination of these drugs in tablets. The results of analysis were validated statistically through recovery studies. Copyright © 2009 John Wiley & Sons, Ltd.
Ethene Polymerization Behavior of MAO‐Activated Dichloridotitanium Complexes Bearing Bi‐ and Tetradentate Salicylaldimine Derivatives
Authors: Pärssinen Antti , Luhtanen Tommi , Pakkanen Tapani , Leskelä Markku , Repo Timo
Published: December 2009

New chiral bridged tetradentate (N2O2)TiIVCl2‐type complexes bearing dimethylbiphenyl (1‐Ti–3‐Ti) and previously published binaphthyl‐bridged (4‐Ti) complex were synthesized with high yields. This was achieved by treating the corresponding Schiff‐base ligand (H2L) precursors with Ti(NMe2)4, followed by conversion of these diamido complexes to LTiCl2 derivatives by the addition of excess of Me3SiCl. A series of unbridged titanium complexes 5‐Ti–8‐Ti with similar substituents at the phenoxy group were studied and their polymerization properties, after methylaluminoxane (MAO) activation, compared with the above bridged complexes. It was found that the catalysts bearing chiral tetradentate biaryl‐bridged salicylaldimine ligands produce multimodal polyethylene (PE) with low activity [below 10 kgPE/(molTi h bar)] while their unbridged analogues provide activities that are 10–1000 times greater under similar reaction conditions. The reasons for this dramatic difference in polymerization activities are discussed based on the stabilities of the different cationic species configurations.
Enantiopure 2‐(Trifluoromethyl)‐1,2,3,4‐tetrahydronaphthalene‐1,2‐diols from a Tartaric Acid Derived Scaffold
Authors: Massicot Fabien , Nonnenmacher Jean , Grellepois Fabienne , Portella Charles
Published: December 2009

The synthesis of the four stereoisomers of 2‐(trifluoromethyl)tetrahydronaphthalene‐1,2‐diols and/or their 2‐O‐allyl derivatives is reported. Nucleophilic trifluoromethylation of a tartaric acid derived keto amide, and a one‐pot hydrolysis/oxidative cleavage/intramolecular Friedel–Crafts transformation are the main features of this synthesis.
Why mood affects help giving, but not moral courage: Comparing two types of prosocial behaviour
Authors: Niesta Kayser Daniela , Greitemeyer Tobias , Fischer Peter , Frey Dieter
Published: December 2009

A long tradition in the help giving literature assumes that mood states determine the level of prosocial behaviour shown by individuals. Most research in this area has been conducted in the context of low cost prosocial behaviour, whereas research has been neglected in which participants were confronted with situations involving potential severe and dangerous negative consequences (i.e., high cost situations) with the help‐giver risking his moral integrity and social disapproval (i.e., moral courage). To address this gap in the literature, the present studies investigate differential effects of positive and negative compared with neutral mood states on help giving versus moral courage. Study 1 shows that in situations requiring low cost helping, participants were more likely to help in positive and negative moods than those in a neutral mood, whereas in situations requiring moral courage (high cost), participants were comparably likely to help in each of the three mood conditions. In Study 2, we find that salience of moral norms mediates the interaction between type of prosocial behaviour and mood. Finally, Study 3 investigates whether the apparent discrepancy between help giving and moral courage as established by the differential impact of mood states can be determined still differently. It reveals that justice sensibility, civil disobedience, resistance to group pressure, moral mandates, and anger lead to moral courage, but not to help giving. Differences between these two types of prosocial behaviour are discussed. Copyright © 2009 John Wiley & Sons, Ltd.
A systematic study in CIEF: Defining and optimizing experimental parameters critical to method reproducibility and robustness
Authors: Mack Scott , Cruzado‐Park Ingrid , Chapman Jeff , Ratnayake Chitra , Vigh Gyula
Published: December 2009

A systematic study of two‐step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Resolution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pI<3) and basic (10<pI) sacrificial ampholyte to the CIEF sample solution. Optimization of the concentrations of the sacrificial ampholytes, iminodiacetic acid and arginine to 1.7 and 40 mM, respectively, yielded a stable focused CA train that spanned the 4<pH<10 range without sacrificing either mAb isoform resolution or sample throughput. Intermediate precision studies performed on the CIEF method with three basic mAbs yielded 0.04 to 0.09% CVs for the estimated pI values and 0.6−3% CVs for isoform group percent composition, indicating that the two‐step CIEF method developed meets the rigorous demands of therapeutic mAb analysis.
A novel methodology for the analysis of membrane and cytosolic sub‐proteomes of erythrocytes by 2‐DE
Authors: Alvarez‐Llamas Gloria , de la Cuesta Fernando , Barderas Maria G. , Darde Veronica M. , Zubiri Irene , Caramelo Carlos , Vivanco Fernando
Published: December 2009

With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, we propose here a novel approach that allows the analysis of both human cytosolic and membrane sub‐proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. We investigate here different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2‐DE, paying particular attention to hemoglobin removal. A simple, quick and satisfactory approach for hemoglobin depletion based on HemogloBind™ reagent was satisfactorily applied to erythrocyte cells, allowing the analysis of the cytosolic sub‐proteome by 2‐DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described here, allowing detection of high molecular weight proteins (i.e. spectrin, ankyrin) and well‐resolved 2‐DE patterns. An aliquot of the membrane fraction was also in solution digested and analyzed by nano‐LC coupled to an LTQ‐Orbitrap mass spectrometer. A total of 188 unique proteins were identified by this approach. This study sets the basis for future clinical studies where the erythrocyte cell may be implicated.
Online CIEF‐ESI‐MS in glycerol–water media with a view to hydrophobic protein applications
Authors: Mokaddem Meriem , Gareil Pierre , Varenne Anne
Published: December 2009

A new online coupling of CIEF with ESI‐MS has been developed in glycerol–water media. This improved protocol provides: (i) the electric continuity during the whole analysis by a discontinuous filling of the capillary with 60:40 (cm/cm) catholyte/proteins–ampholyte mixture; (ii) the use of an anticonvective medium, i.e. 30:70 glycerol/water, v/v, compatible with MS detection and as an aid to hydrophobic protein solubilization and (iii) the use of unmodified bare fused‐silica capillaries, as the glycerol/water medium strongly reduces EOF. Focusing was performed in positive polarity and cathodic mobilization was achieved by both voltage and pressure application. The setup was optimized with respect to analysis time, sensitivity and precision on pI determination. The optimized anolyte and catholyte were composed of 50 mM formic acid/1 mM glutamic acid (pH 2.35) and 100 mM NH3/1 mM lysine (pH 10.6), respectively. The effects of ampholyte concentration, focusing time and ESI parameters were presented for model proteins and discussed. This new integrated protocol should be an easy and effective additional tool in the field of proteome analysis, providing a means for the characterization of a large number of hydrophilic and hydrophobic proteins.
Microchip isoelectric focusing with monolithic immobilized pH gradient materials for proteins separation
Authors: Liang Yu , Cong Yongzheng , Liang Zhen , Zhang Lihua , Zhang Yukui
Published: December 2009

Monolithic immobilized pH gradient (M‐IPG) materials were prepared in microchannles by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by the attachment of focused Ampholine onto the surface of porous monoliths via epoxide groups. With M‐IPG materials as matrix, FITC‐labeled ribonuclease B, myoglobin and α‐casein were well separated by microchip isoelectric focusing (μCIEF) without carrier amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were applied to drive focused zones for LIF detection. Our experimental results showed that pressure mobilization was preferable with neglectable band broadening, and good peak shape and high detection sensitivity were obtained. All these results demonstrate that μCIEF with M‐IPG materials is not only an efficient mode for protein enrichment and separation but also attractive to couple with other CE modes to achieve multi‐dimensional separation or MS for further identification, without the interference of mobile CAs.
Study of metallothionein oxidation by using of chip CE
Authors: Krizkova Sona , Adam Vojtech , Kizek Rene
Published: December 2009

Metallothionein (MT) is a low‐molecular mass protein playing an essential role in homeostasis of heavy metals ions. We used a chip‐based CE for quantitative study of MT oxidation. After oxidation of MT by H2O2 we observed marked decrease in peaks heights and shift of peaks positions to higher molecular mass, which corresponded with the time of the oxidation. Moreover, we observed that the proportion of high‐molecular forms of MT was markedly increased. The oxidative changes were successfully reversed by using of reducing agent prior to electrophoresis. Our method can reveal the stability MT aggregates, of which biological role still remains unclear.
Quantitative protein expression profiling of 14‐3‐3 isoforms in human renal carcinoma shows 14‐3‐3 epsilon is involved in limitedly increasing renal cell proliferation
Authors: Liang Shufang , Xu Yuhuan , Shen Guobo , Liu Qingping , Zhao Xinyu , Xu Zhizhong , Xie Xi , Gong Fengming , Li Ronghui , Wei Yuquan
Published: December 2009

14‐3‐3 proteins regulate many cellular processes that are implicated in cancer development, and the seven 14‐3‐3 isoforms have different expression level and isoform‐specific roles in different tumors. However, the biological functions of 14‐3‐3 proteins and their correlations with renal carcinoma have not been investigated so far. In our study, the expression profiles and functional characterization of 14‐3‐3 proteins were discovered by a sensitive stable isotope labeling with amino acids in cell culture based quantitative proteomics analysis in human renal carcinoma tissues. We found that 14‐3‐3ε was up‐regulated with 1.44‐fold changes in renal cancerous tissues compared with that in counterpart kidney tissues, and 14‐3‐3σ was almost not detected in both tissues due to its DNA highly methylated in our previous reports. The other five isoforms almost have similar expression level in two states of renal tissues. The following RT‐PCR, Western blot and immunohistochemistry analysis for specific 14‐3‐3 isoform expression were all consistent with the quantitative proteomic data. Furthermore, the overexpression of 14‐3‐3ε in vitro can limitedly prompt the abnormal growth of renal tumor cells.
Robust features for 2‐DE gel image registration
Authors: Möller Birgit , Posch Stefan
Published: December 2009

Proteomics is a rapidly growing field of modern biology. Since quantitative data of proteins involved in dynamic processes of living organisms are essential for understanding the basics of life, techniques like 2‐DE and related procedures for automatic data interpretation are at the heart of this research field. They are strongly required to enable analysis and interpretation of the emerging amount of available data. Analyzing and interpreting gel image data usually requires the comparison of gels from different experiments and, thus, a prior registration of gels. This can be accomplished using featureless, feature‐based or hybrid registration approaches combining both techniques. Recently, the latter ones have shown high performance, and it is undoubtful that in general robust and reliable features are an essential ingredient and valuable source of information for high‐quality image registration. In this paper we provide a thorough overview and elaborate analysis of the capabilities of available feature detectors for gel image registration. Particularly, a detailed and extensive comparative study is presented where common spot‐specific detectors are included as well as image‐content independent detectors that were not applied to the task of gel image registration until now. The study incorporates tests on several thousand synthetically deformed images from different experimental conditions. As a result it provides valuable quantitative data allowing for direct objective comparisons of various detectors, and is well suited to guide the design of new registration algorithms.
Analysis of integral membrane proteins by heat gel‐embedment combined with improved in‐gel digestions
Authors: Zhou Jian , Huang Sha , Bi Deng , Zhang Hai , Li Jianglin , Lin Yong , Chen Ping , Wang Xianchun , Liang Songping
Published: December 2009

Analysis of integral membrane proteins (IMPs) presents a special challenge because of their hydrophobic nature and low abundance. Here, a new method was developed, which involved heat gel‐embedment and improved in‐gel digestion of the proteins. Membrane protein lysate containing detergents was mixed with acrylamide solution and the proteins were embedded when the gel polymerized. For comparison, the protein embedment was made at different temperatures (25, 35 or 45°C), and the in‐gel digestions were performed in the presence of 0.1% RapiGest reagent (ALS), 0.1% sodium deoxycholate and 10% ACN, respectively. The resultant peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry. Compared with that at 25°C, gel‐embedment at 45°C improved the protein embedment and thus protein identification, with the identified IMPs increased by 27%. 0.1% sodium deoxycholate was more efficient than 0.1% ALS and 10% ACN in terms of improving the digestion and tryptic digest recovery of the gel‐embedded proteins particularly the hydrophobic IMPs. Out of the 326 IMPs identified by heat gel‐embedment combined with improved in‐gel digestion strategies, 149 (46%) proteins had at least two mapped transmembrane domains. These results indicate that our newly developed protocol could facilitate the high throughput analysis of integral membrane proteome.
CE‐MALDI interface based on inkjet technology
Authors: Vannatta Michael W. , Whitmore Colin D. , Dovichi Norman J.
Published: December 2009

An ink jet printer valve and a nozzle were used to deliver matrix and sample from an electrophoresis capillary onto a MALDI plate. The system was evaluated by the separation of a set of standard peptides. That separation generated up to 40 000 theoretical plates in less than 3 min. Detection limits were 500 amol for an ABI TOF‐TOF instrument and 2 fmol for an ABI Q‐TOF instrument. Over 70% coverage was obtained for the tryptic digest of α‐lactalbumin in less than 2.5 min.
Optimisation of protein extraction and 2‐DE for metaproteomics of microbial communities from anaerobic wastewater treatment biofilms
Authors: Abram Florence , Gunnigle Eoin , O'Flaherty Vincent
Published: December 2009

The feasibility of metaproteomic analysis of the microbial communities underpinning anaerobic wastewater treatment relies on efficient protein extraction and separation techniques. The microorganisms involved in anaerobic digestion (AD) of wastewater typically aggregate to form tightly organised, spherical granules, from which protein extraction is challenging. Here, we compare two methods of protein extraction, one using a French press previously used successfully to analyse the proteome of an activated sludge [Wilmes, P., Bond, P. L., Environ. Microbiol. 2004, 6, 911–920.] and one using sonication developed in the context of pure culture [Abram, F., Wan‐Lin, S., Wiedmann, M., Boor, K. J., Coote, P., Botting, C., Karatzas, K. A. G., O'Byrne, C. P., Appl. Environ. Microbiol. 2008, 74, 594–604.]. We used 2‐DE to carry out this comparison. The protein extraction using the sonication method resulted in a significant increase in the number of protein spots and higher quality 2‐D gels.
Cognitive impairment and the role of the ApoE ε4‐allele after stroke—a 13 months follow‐up study
Authors: Wagle Jørgen , Farner Lasse , Flekkøy Kjell , Wyller Torgeir B. , Sandvik Leiv , Eiklid Kristin L. , Fure Brynjar , Stensrød Brynhild , Engedal Knut
Published: December 2009

Objective

To examine the relationship between the ApoE ε4 allele and cognitive impairment 13 months after stroke.

Methods

One hundred four stroke rehabilitation patients were cognitively tested on average 18 days after hospital admission and again 13 months later with the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). The following potential risk factors for post‐stroke cognitive impairment (defined by a RBANS total index score below 77.5 points) at 13 months follow‐up were analyzed in bivariate and logistic regression analyses: ApoE‐genotype, socio‐demographic variables, pre‐stroke cognitive reduction (The Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE)), vascular factors, lesion characteristics, and neurological impairment (The National Institute of Health Stroke Scale (NIHSS)). Differences in general cognitive performance (pre‐stroke, baseline, and follow‐up) across patients with different ApoE‐genotypes were analyzed, and lastly differences between ε4‐carriers and non‐carriers for changes in performance in various cognitive domains over the 13 months period were examined.

Results

Significant risk factors for cognitive impairment at 13 months were ApoE ε4, pre‐stroke cognitive reduction (IQCODE 3.44+), previous stroke, and neurological impairment (NIHSS Total Score >5). A significant dose‐dependent effect of the ApoE‐genotype in relation to overall post‐stroke cognitive functioning was found at baseline and follow‐up, but not pre‐stroke. The ε4 carriers showed a significant decline in tests related to verbal learning and memory compared to the non‐carriers.

Conclusions

The ApoE ε4‐allele constitutes an independent risk factor for cognitive impairment at 13 months post‐stroke, and is associated with progression of cognitive decline in tasks related to verbal learning and memory. Copyright © 2009 John Wiley & Sons, Ltd.
Human papillomavirus testing and cervical cytology in primary screening for cervical cancer among women in rural China: Comparison of sensitivity, specificity, and frequency of referral
Authors: Moy Lisa M. , Zhao Fang‐Hui , Li Long‐Yu , Ma Jun‐Fei , Zhang Qing‐Ming , Chen Feng , Song Yan , Hu Shang‐Ying , Balasubramanian Akhila , Pan Qin‐Jing , Koutsky Laura , Zhang Wen‐Hua , Lim Jeanette M. , Qiao You‐Lin , Sellors John W.
Published: December 2009

The causal relationship between persistent high‐risk human papillomavirus infection and cervical cancer is widely accepted. HR‐HPV DNA testing, alone or in combination with Pap smear testing, may have a role in primary screening. The screening results (VIA, VILI, Pap, and HR‐HPV DNA) of 9,057 women in rural China were analyzed to determine the screening performance for the detection of CIN3+. All screening strategies had comparable AUCs (0.9). Cotesting strategies had the overall highest sensitivity for CIN3+ (99.4%), followed by HR‐HPV DNA testing alone (96.3%), Pap alone (80.2%), and reflex testing (75.4%). Reflex testing had the highest specificity (96.7%), followed by Pap alone (93.3%), HR‐HPV DNA testing alone (85.5%), and both cotesting strategies (LSIL: 84.8%, HSIL: 84.8%). Of the single‐test strategies, HR‐HPV DNA testing had a higher sensitivity (96.3% vs. 80.2%) compared with Pap testing. The specificity of the Pap test was higher (93.3% vs. 85.5%) and it had a lower percent referred for colposcopy (7.8% vs. 15.8%) than HR‐HPV DNA testing. HR‐HPV DNA testing with a 10.0 cutoff point (relative light units/cutoff ratio) had a sensitivity (85.2%) and specificity (90.6%) estimate comparable to Pap testing. A single‐test primary screening strategy with adequate performance would permit less frequent screening and be most appropriate. Of the primary screening strategies investigated in this setting in China, the performance of HR‐HPV DNA testing with an increased cutoff‐point might best meet these criteria.
Imaging of branched chain amino acid metabolism in tumors with hyperpolarized 13C ketoisocaproate
Authors: Karlsson Magnus , Jensen Pernille R. , in 't Zandt René , Gisselsson Anna , Hansson Georg , Duus Jens Ø. , Meier Sebastian , Lerche Mathilde H.
Published: December 2009

Powerful analytical tools are vital for characterizing the complex molecular changes underlying oncogenesis and cancer treatment. This is particularly true, if information is to be collected in vivo by noninvasive approaches. In the recent past, hyperpolarized 13C magnetic resonance (MR) spectroscopy has been employed to quickly collect detailed spectral information on the chemical fate of tracer molecules in different tissues at high sensitivity. Here, we report a preclinical study showing that α‐ketoisocaproic acid (KIC) can be used to assess molecular signatures of tumors with hyperpolarized MR spectroscopy. KIC is metabolized to leucine by the enzyme branched chain amino acid transferase (BCAT), which is found upregulated in some tumors. BCAT is a putative marker for metastasis and a target of the proto‐oncogene c‐myc. Very different fluxes through the BCAT‐catalyzed reaction can be detected for murine lymphoma (EL4) and rat mammary adenocarcinoma (R3230AC) tumors in vivo. EL4 tumors show a more than 7‐fold higher hyperpolarized 13C leucine signal relative to the surrounding healthy tissue. In R3230AC tumor on the other hand branched chain amino acid metabolism is not enhanced relative to surrounding tissues. The distinct molecular signatures of branched chain amino acid metabolism in EL4 and R3230AC tumors correlate well with ex vivo assays of BCAT activity.
Targeting endothelin A receptor enhances anti‐proliferative and anti‐invasive effects of the HER2 antibody trastuzumab in HER2‐overexpressing breast cancer cells
Authors: Fischgräbe Jeanett , Götte Martin , Michels Katharina , Kiesel Ludwig , Wülfing Pia
Published: December 2009

Human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in human breast cancers. It is known to drive tumor growth and progression and represents a prominent target in breast cancer therapy. The endothelin (ET) system, in particular ET‐1 and its receptor ETAR, is of major relevance for breast cancer growth and invasion. Having previously demonstrated coexpression of ETAR and HER2 in breast tumors, this study was designed to investigate molecular interactions of HER2 (including the epidermal growth factor receptor EGFR as its major coreceptor) and ET signaling, and the potential benefit of a combined anti‐HER2/ETAR treatment in human breast cancer cells. Dual HER2‐ETAR targeting utilizing trastuzumab (monoclonal anti‐HER2 antibody) and the ETAR antagonist atrasentan was superior to each agent alone in inhibiting basal and EGF‐induced proliferation and invasion of HER2‐overexpressing BT‐474 and SK‐BR‐3 cells. EGF‐induced invasion was partially inhibited by atrasentan alone, suggesting the involvement of ETAR in EGF receptor mediated invasion of breast cancer cells. Moreover, secretion of the pro‐invasive ET‐1 was shown to be induced by EGF via EGFR and HER2, including MAPK‐dependent signaling. In turn, an ET‐1/ETAR‐dependent regulation of EGFR protein expression and phosphorylation (at Tyr845) was observed, which may contribute to the additional anti‐proliferative and anti‐invasive effects of atrasentan on trastuzumab treated cells; reconfirming, atrasentan failed to enhance inhibitory effects of EGFR‐targeted agents. This study suggests complex interactions between HER2/EGFR and ET pathways in breast cancer and supports the hypothesis that dual HER2‐ETAR targeting may represent a highly effective approach in breast cancer treatment.
Circulating free DNA, p53 antibody and mutations of KRAS gene in endometrial cancer
Authors: Dobrzycka Bozena , Terlikowski Slawomir J. , Mazurek Andrzej , Kowalczuk Oksana , Niklinska Wieslawa , Chyczewski Lech , Kulikowski Marek
Published: December 2009

This study was conducted to evaluate the significance of circulating free DNA (CFDNA), p53 antibody (p53‐Ab) and mutations of KRAS gene in the development of endometrial cancer (EC). A total of 109 patients with EC (87 patients with Type I and 22 patients with Type II) took part in this study. KRAS mutations and CFDNA were detected by means of the PCR‐RFLP and enriched by the PCR‐RFPL method. ELISA was used to analyze plasma p53‐Ab. Tissue expression of P53 protein was evaluated immunohistochemically (IHC). The frequency of KRAS mutations was especially high in Grade 2 of Type I EC. CFDNA was frequently detected in patients with early stage of Type II EC at a low level of grade. It is noteworthy that the p53‐Ab positive rate increased in the higher grade of Type I tumors. A significant difference in the number of cases with the p53‐Ab was found in the advanced stage of Type I tumors. The frequency of KRAS and p53‐Ab correlates with tumor stage only in the Type I EC. Plasma CFDNA and p53‐Ab offer a chance to develop a procedure for EC Type II diagnosis. The association between tumor cells related to CFDNA and p53‐Ab with Type II tumor suggests that it might potentially serve as a marker in predicting the prognosis and offers a possibility to individualize treatment regimen.
Type‐specific human papillomavirus oncogene messenger RNA levels correlate with the severity of cervical neoplasia
Authors: Ho Chih‐Ming , Lee Bor‐Heng , Chang Shwu‐Fen , Chien Tsai‐Yen , Huang Shih‐Hung , Yan Chiu‐Cho , Cheng Wen‐Fang
Published: December 2009

This study aimed to evaluate whether quantitation of high‐risk human papillomavirus (HR‐HPV) E6 messenger RNA (mRNA) can be a potential biomarker for detecting the severity of cervical lesions. HPV genotyping was performed using a modified MY11/GP6+ PCR for HPV DNA amplification, followed by HPV genotype‐specific hybridization with on a gene chip. E6 type‐specific PCR was used to validate multiple infections. Quantitative real‐time reverse transcriptase (QRT‐PCR) and real‐time PCR used to measure mRNA levels and DNA viral loads of 6 HPV oncogenic types (HPV 16, 18, 31, 33, 52 and 58) in 720 liquid‐based cytology samples. The HPV DNA and RNA measurements were correlated with cervical lesions diagnosed by histopathologic examination. mRNA transcripts in the 6 types HPV DNA‐positive cases was lower in normal women and <CIN 1 (23%), women with CIN 1 (54%), CIN2+ (77%) and CIN3+ (80%) (p < 0.001). Geometric mean mRNA levels ranged from 24.5 (copies per 50 ng total RNA) in normal women and <CIN 1 to 210.8 in those with CIN 1, 629.0 in CIN2+ and 699.0 in CIN3+ (p < 0.0001). Trends of increasing viral mRNA with severity of histopathologic diagnosis were significant for HPV 16, 18, 52 and 58 transcripts but not for HPV 31 and 33 transcripts. However, geometric mean DNA viral loads of HPV 16, 18, 52 and 58 DNA did not significantly increase with the severity of cervical dysplasia. Therefore, quantitative HPV E6 mRNA levels of high‐risk HPV types are potentially useful biomarkers for distinguishing among HPV infections, cervical precancerous lesions and cancer.
Phosphatase of regenerating liver‐3 as a convergent therapeutic target for lymph node metastasis in esophageal squamous cell carcinoma
Authors: Ooki Akira , Yamashita Keishi , Kikuchi Shiro , Sakuramoto Shinichi , Katada Natsuya , Watanabe Masahiko
Published: December 2009

Phosphatase of regenerating liver‐3 (PRL‐3) is a molecule associated with metastasis in a diverse of cancers, which, however, remains largely unknown in esophageal squamous cell carcinoma (ESCC). We examined both the clinical significance of PRL‐3 expression and its biological roles, and assessed possibilities as a therapeutic target in ESCC. PRL‐3 expression was found in 78% (69 of 88) of the primary ESCC on immunohistochemistry; it was the strong independent predictor for lymph node metastasis (LNM) on a multivariate logistic regression model (p = 0.0014, relative risk =15.20). Additionally, gene amplification was found in 3 (7.9%) of the 38 primary tumors with PRL‐3 overexpression by fluorescence in situ hybridization, but in none of the 19 tumors without it. PRL‐3 small interfering RNA robustly repressed cell proliferation, anchorage‐independent colony formation and invasion and augmented 5‐FU‐induced apoptosis in all the tested ESCC cell lines with PRL‐3 overexpression, irrespective of its gene amplification status. PRL‐3 inhibitor (1‐4‐bromo‐2‐benzylidene rhodanine) also suppressed such metastatic properties in the cell lines with PRL‐3 overexpression, but not with its low expression. Inverse effects were observed by PRL‐3 forced expression. Collectively, PRL‐3 overexpression is a frequent event associated with LNM and plays a causative role in promoting cancer progression. Moreover, the expression status may be a landmark to select patients with benefit from PRL‐3‐targeted therapy. Thus, PRL‐3 could be a convergent therapeutic target against ESCC with LNM.
Protein expression profiling of primary mammary epithelial cells derived from MMTV‐neu mice revealed that HER2/NEU‐driven changes in protein expression are functionally clustered
Authors: Park Sungwoo , Lee Kyung‐min , Ju Ji‐hyun , Kim Jaeyoon , Noh Dong‐Young , Lee Taehoon , Shin Incheol
Published: December 2009

MMTV‐neu transgenic mice overexpressing NEU in their mammary glands develop tumor after 6 months of age. To find a novel protein biomarker using this mouse model, we identified and characterized the proteins that were differently expressed between primary mammary epithelial cells from 2 months old MMTV‐neu heterozygote mice and wild type (WT) littermates using two‐dimensional digest (ChemDigest™/Trypsin)‐LC‐MS/MS. The differentially expressed proteins were selected and analyzed using DAVID Bioinformatics resource. The proteins involved in anti‐apoptosis, purine metabolism, ribosome and proteasome functions were upregulated, whereas cell adhesion‐related proteins were downregulated in PMECs from MMTV‐neu mice when compared with WT PMECs. The results indicate that several functional units are coregulated by HER2/NEU. We hypothesize that these changes in the cellular proteome may be responsible for early onset of HER2/NEU‐driven tumorigenesis. © 2009 IUBMB IUBMB Life, 62(1):41–50, 2010
Caveolin‐1 facilitates cyclooxygenase‐2 protein degradation
Authors: Chen Shu‐Fen , Liou Jun‐Yang , Huang Tai‐Yu , Lin Yu‐Sin , Yeh Ai‐Ling , Tam Kabik , Tsai Tsung‐Huang , Wu Kenneth K. , Shyue Song‐Kun
Published: December 2009

Cyclooxygenase‐2 (COX‐2) plays major roles in diverse physiological and pathological processes such as inflammation and tumorigenesis. Transcriptional control of COX‐2 has been extensively investigated and characterized, but its post‐translational control is less clear. Here, we report a novel mechanism by which COX‐2 is degraded. Protein levels of caveolin‐1 (Cav‐1) and COX‐2 showed an inverse relation in colon cancer cell lines. COX‐2 proteins in lung and colon tissues were higher in Cav‐1 null mice than in wild‐type mice. RNAi knockdown of Cav‐1 increased COX‐2 protein level and decreased ubiquitinated COX‐2 accumulation. In addition, deletion of the carboxy (C)‐terminus of COX‐2, which contains a unique 19‐amino acid segment compared with COX‐1, resulted in reduced Cav‐1 binding and attenuated COX‐2 degradation. COX‐1 and green fluorescence protein containing the C‐terminus of COX‐2 resulted in enhanced degradation. Our findings suggest that Cav‐1 binds COX‐2 in endoplasmic reticulum (ER) and carries it for degradation via ER associated degradation. The C‐terminal region of COX‐2 is required for Cav‐1 binding and degradation. These results indicate a novel function of Cav‐1 in controlling COX‐2 expression, which may regulate physiological functions and have tumor suppression effects. J. Cell. Biochem. 109: 356–362, 2010. © 2009 Wiley‐Liss, Inc.
Purification and characterization of active caspase‐14 from human epidermis and development of the cleavage site‐directed antibody
Authors: Hibino Toshihiko , Fujita Eriko , Tsuji Yumiko , Nakanishi Jotaro , Iwaki Haruhi , Katagiri Chika , Momoi Takashi
Published: December 2009

Restricted expression of caspase‐14 in differentiating keratinocytes suggests the involvement of caspase‐14 in terminal differentiation. We purified active caspase‐14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764‐fold with a yield of 9.1%. Purified caspase‐14 revealed the highest activity on WEHD‐methylcoumaryl‐amide (MCA), although YVAD‐MCA, another caspase‐1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N‐terminal and C‐terminal analyses demonstrated that the large subunit consisted of Ser6‐Asp146 and N‐terminal of small subunit was identified as Lys153. We successfully developed an antiserum (anti‐h14D146) directed against the Asp146 cleavage site, which reacted only with active caspase‐14 but not with procaspase‐14. Furthermore we confirmed that anti‐h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti‐h14D146 staining was mostly restricted to the cornified layer and co‐localized with some of the TUNEL positive‐granular cells in the normal human epidermis. UV radiation study demonstrated that caspase‐3 was activated and co‐localized with TUNEL‐positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase‐14 activation in response to UV. Our study revealed tightly regulated action of caspase‐14, in which only the terminal differentiation of keratinocytes controls its activation process. J. Cell. Biochem. 109: 487–497, 2010. © 2009 Wiley‐Liss, Inc.
IR‐spektroskopische Untersuchung von 1:1‐Addukten aus Bortrihalogeniden und Acylaziden
Authors: Fahr Egon , Neumann Lutz
Published: December 2009

Bei der Umsetzung von Acylaziden 5 mit BF3 bei ca. −60° entstehen sehr zersetzliche 1:1‐Addukte 6, die in einigen Fällen analysenrein erhalten werden konnten. Ihre IR‐spektroskopische Untersuchung zeigt, daß die Addukt‐Bildung durch Angriff des Bortrihalogenids an der CO‐Gruppierung des Acylazids erfolgt.
Acyl‐lacton‐Umlagerung, XLIV1) NMR‐spektroskopische Untersuchungen an α‐Acyl‐lactonen,‐lactamen bzw. ‐thiollactonen und Bestimmung der Keto‐Enol‐Gleichgewichte
Authors: Wamhoff Heinrich , Lander Helmut , Korte Friedhelm
Published: December 2009

Aus den NMR‐Spektren von 22 α‐Acyl‐lactonen, ‐lactamen und ‐thiollactonen in Deuterochloroform und Hexadeutero‐dimethylsulfoxid werden die jeweiligen Keto‐Enol‐Gleichgewichte in Abhängigkeit von Acyl‐Rest und Substitution bestimmt. Sie stimmen in nahezu allen Fällen mit den durch Enol‐Titration gewonnenen Werten überein.
Über die Struktur der Thioamide und ihrer Derivate, II1) Untersuchungen an N‐[β‐Hydroxy‐äthyl]‐N‐alkyl‐thioformamiden. Trennung von geometrischen Isomeren und NMR‐spektroskopische Konfigurationszuordnungen
Authors: Walter Wolfgang , Maerten Gerhard
Published: December 2009

N[β‐Hydroxy‐äthyl‐]N‐alkyl‐thioformamide mit den Resten Methyl (1), Äthyl (2) und Isopropyl (3) werden durch präparative Schichtchromatographie in geometrische Isomere (a bzw. b) getrennt und deren Konfigurationen durch NMR‐Spektroskopie bestimmt. In den Gleichgewichten überwiegt die Konfiguration, in welcher der größere Substituent dem Schwefel gegenübersteht. Die N‐tert.‐Butyl‐Verbindung 4 ist chromatographisch einheitlich. An der N‐Äthyl‐Verbindung 2 werden die Enthalpie‐Differenz der Isomeren (ΔH = 610 ± 150 cal/mol) und bei 60° die freie Aktivierungsenthalpie der behinderten Rotation (25.2 ± 0.5 kcal/Mol) bestimmt.
Lewissäure‐katalysierte Umsetzung von aliphatischen Ketonen mit tert.‐Butyl‐isonitril
Authors: Zeeh Bernd , Müller Eugen
Published: December 2009

Aus Aceton bzw. Methyl‐äthyl‐keton und tert.‐Butyl‐isonitril bilden sich in Anwesenheit von Bortrifluorid Alken‐(1)‐yl‐(2)‐glyoxylsäure‐tert.‐butylamide (2 bzw. 3) sowie α‐Hydroxycarbonsäure‐tert.‐butylamide (7 bzw. 8).
Selen‐spirane
Authors: Hellwinkel Dieter , Fahrbach Gerhard
Published: December 2009

Bei der Umsetzung von 2.2′‐Biphenylylen‐selenid‐tosylimin (7a) mit 2.2′‐Dilithium‐biphenyl (8) entsteht Bis‐2.′‐biphenylylen‐selen (4a), das mit Wasser bzw. Brom unter Öffnung eines Ringes zu den Selenoniumsalzen 9 bzw. 10 reagiert. Analog erhält man Bis‐4.4′‐dimethyl‐2.2′‐biphenylylen‐selen (4b), das, wie u. a. aus massenspektroskopischen Untersuchungen folgt, wesentlich instabiler als die Stammverbindung 4a, ist.
Synthesen mit substituierten Malondialdehyden, IV1) γ‐Nitro‐tetramethinmerocyanin‐ und γ‐Nitro‐pentamethincyanin‐Farbstoffe2)
Authors: Reichardt Christian
Published: December 2009

Durch Kondensation von Natrium‐nitromalondialdehyd (2) mit heterocyclischen Immoniumsalzen 1 werden neben Tetramethinmerocyanin‐Farbstoffen 3 symmetrische und unsymmetrische Pentamethincyanin‐Farbstoffe 4 dargestellt, deren Polymethin‐Kette durch eine Nitrogruppe substituiert ist. Diese Farbstoffe eignen sich zum Studium der Beziehung zwischen der Elektronegativität eines Substituenten und seinem Einfluß auf das UV‐Absorptionsspektrum (Abb. 4).
Synthese von Azastilbenen
Authors: Zymalkowski Felix , Reimann Eberhard
Published: December 2009

Zur Synthese N‐haltiger Analoga des Stilböstrols werden aus 4‐Acyl‐pyridinen (4, 9, 12) mit Lithium‐organischen Verbindungen die entsprechenden tertiären Alkohole (10, 13) gewonnen. Diese liefern durch Wasser‐Abspaltung bei tiefer Temperatur Azastilbene (2, 14) als cis/trans‐Isomeren‐Gemische. Ihre Stilben‐Struktur wird durch das NMR‐Spektrum bewiesen.
Über ein Imidazolium‐Analogon des Thiamins mit antagonistischer Wirkung
Authors: Staab Heinz A. , Schwalbach Gerd A.
Published: December 2009

Eine Thiamin‐analoge Verbindung (2), in der der Thiazolium‐Teil des Thiamins durch ein entsprechend substituierte N‐Methyl‐imidazolium‐Gruppe ersetzt ist, wird dargestellt und erweist sich bei Lactobacillus fermenti als Thiamin‐Antagonist.
Synthesen in der Reihe der Indole und Indolalkaloide, VII1) Synthese eines undecacyclischen Di‐indolin‐diäthers vom Typ dimerer Calebassencurare‐Alkaloide
Authors: Fritz Helmut , Rubach Günther
Published: December 2009

Die Synthese des diquartären Ammoniumsalzes 4 wird beschrieben. Diese undecacyclische Verbindung hat die wesentlichen Konstitutionsmerkmale dimerer Calebassencurare‐Alkaloide, die in ihrem zentralen Ringsystem auf der Oxydationsstufe von C‐Curarin‐I stehen. – Im Carbazolenin 9 und der dimeren Verbindung 19 sind zwei Urethane neuen Typs aufgefunden worden, in deren NMR‐Spektren die O‐Äthyl‐Signale eine temperaturabhängige Aufspaltung zeigen.
Ubichinone und verwandte Substanzen, XII1) Umsetzung von Acetyl‐ubichromenol(6) mit Lithiumalanat
Authors: Morimoto Hiroshi , Imada Isuke , Watanabe Masazumi , Sugihara Hirosada
Published: December 2009

Aus Acetyl‐ubichromenol(6) (3) werden mit LiAlH4 γ‐Hydroxy‐isoubichinon (7) (4, γ‐Hydroxy‐β.γ‐dihydro‐ubichinon(7) (5), Ubichinon(7) (6), Desmethyl‐ubichinon(7) (7; 2‐Methoxy‐3‐hydroxy‐5‐wird dadurch nachgewiesen, daß man Ubichromenol(6) (2) mit Natrium in Äthanol zu Ubichromanol(6) (8) reduziert und dieses oxydativ zu 5 aufspaltet. Aus den NMR‐Spektren inst zu schließen, daß 6 und 7 aus einem Gemisch der cis‐trans‐Isomeren an der Δβ‐Doppelbindung in der Isopren‐Seitenkette bestehen. Die Mechanismen dieser Reaktionen werden mit Hilfe von LiAlD4 (statt LiAlH4) und anhand der Massenspektren studiert.
Chemie und Biochemie der Pflanzenstoffe, XXII1) 6‐ und 7‐substituierte 1.4‐Dioxo‐isochromane
Authors: Reichel Ludwig , Hampel Winfried
Published: December 2009

6‐ und 7‐substituierte 1.4‐Dioxo‐isochromane (4a – f) werden aus 4‐substituierten Phthalsäureanhydriden (1a‐‐f) über die Monomethylester. Säurechloride und Diazomethylketone dargestellt. Die Reaktion von 4‐Halogen‐phthalsäureanhydriden mit absol. Methanol führt bevorzugt zu 2‐Methoxycarbonyl‐4‐halogen‐benzoesäuren (5a–d). 4‐Halogen‐homophthalsäuren (9a und b) b) werden synthetisiert und zum Konstitutionsbeweis von 5a – herangezogen. 4‐Methoxy‐phthalsäureanhydrid (1e) liefert mit absol. Methanol ebenfalls überwiegend den 2‐Monomethylester 5e.
Zur Kenntnis der Proanthocyanidine, XI1) Konfigurationsbestimmung von C30H26O12‐Procyanidinen und Strukturaufklärung eines neuen Procyanidins
Authors: Weinges Klaus , Göritz Klaus , Nader Franz
Published: December 2009

Mit Hilfe der NMR‐Spektren werden 4 diastereomeren Decaacetyl‐procyanidinen B1–B4 die Konfigurationen 4–7 zugeordnet. Ein Procyanidin der Summenformel C30H26O11 wird aus den Hülsen der Glyzinie (Wistaria sinensis) isoliert; seine Konstitution 2 und die Konfiguration 8 seines Nonaacetats ergeben sich massenspektrometrisch und durch Vergleich mit dem NMR‐Spektrum von 7.
Zuckerperoxide IV1) Darstellung, Reaktionen und NMR‐Untersuchungen von Zucker‐1.2‐peroxyorthoestern2)
Authors: Schulz Manfred , Boeden Hans‐Friedrich , Berlin Peter , Bley Wolf‐Rüdiger
Published: December 2009

Die Synthese der acetylierten 1.2‐tert.‐Butylperoxyrothoacetate von D‐Glucose, D‐Galaktose, D‐Mannose, L‐Rhamnose und D‐Xylose sowie der benzoylierten 1.2‐tert.‐Butylperoxyorthobenzoate von pyranoider und furanoider L‐Arabinose wird beschrieben (Tab. 1). Die durch Entacetylierung der Verbindungen erhaltenen Zucker‐1.2.‐tert.‐butyl‐peroxyrothoester (Tab. 2) mit freien Hydroxylgruppen sind kristallin. Mit wasserfreien Säuren lagern sich die acylierten tert.‐Butylperoxyorthoester zu acylierten Peroxyglykosiden um (Tab. 3). Aus den Ergebnissen von NMR‐Untersuchungen der acylierten Zucker‐1.2‐tert.‐butylperoxyorthoester werden mit den Kopplungskonstanten (Tab. 4) unter Voraussetzung der Gültigkeit der Karplus‐Beziehung und mit Hilfe von Dreiding‐Modellen Konformationen abgeleitet: für das D‐Glucose‐ und das D‐Xylose‐Derivat (3 bzw. 7) einen verzerrte Wanne, für das D‐Galaktose‐,D‐Mannose‐ bzw. L‐Rhamnose‐Derivat (5 bzw. 11 bzw. 13) ein verzerrter Sessel.