bluuebunny/crossref_metadata_2025 huggingface dataset:
KIT (CD117) is frequently overexpressed in thymic carcinomas but is absent in thymomas
Authors: Pan Chin‐Chen , Chen Paul Chih‐Hsueh , Chiang Hung
Published: February 2004

KIT (CD117), a tyrosine kinase receptor, has not been widely studied in epithelial tumours. In a systematic immunohistochemical survey of KIT expression on tissue arrays incorporating 671 cases, it was found that thymic carcinomas frequently express KIT. Twenty‐two thymic carcinomas, 110 thymomas, and 16 non‐neoplastic thymus glands were retrieved for further analyses. Immunohistochemically, 19 (86%) thymic carcinomas revealed heterogeneous to diffuse membranous positivity, whereas no thymomas or normal thymus glands contained positive epithelial cells. Using reverse transcriptase‐polymerase chain reaction (RT‐PCR), c‐kit transcripts could be demonstrated in all immunohistochemically positive cases. PCR amplification and direct sequencing of the c‐kit juxtamembrane domains (exons 9 and 11) and tyrosine kinase domain (exons 13 and 17) were also performed on the thymic carcinomas but mutations were not found. Some non‐thymic epithelial tumours showed frequent KIT expression including adenoid cystic carcinomas of the salivary gland (100% positive), chromophobe renal cell carcinomas (94%), renal oncocytomas (67%), and neuroendocrine tumours (34%). Other carcinomas were infrequently immunoreactive for KIT. The findings of this study suggest that KIT is involved in the pathogenesis of thymic carcinomas. The overexpression of KIT in thymic carcinomas has potential diagnostic utility in differentiating these tumours from thymomas and carcinomas arising from other sites, which express KIT infrequently. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Molecular analysis of the INK4A/INK4A‐ARF gene locus in conventional (central) chondrosarcomas and enchondromas: indication of an important gene for tumour progression
Authors: van Beerendonk Hetty M , Rozeman Leida B , Taminiau Antonie HM , Sciot Raf , Bovée Judith VMG , Cleton‐Jansen Anne‐Marie , Hogendoorn Pancras CW
Published: February 2004

Loss of heterozygosity (LOH) at chromosomal band 9p21 is one of the few consistent genetic aberrations found in conventional chondrosarcoma. This locus harbours two cell‐cycle regulators, CDKN2A/p16/INK4A and INK4A‐p14ARF, which are inactivated in various human malignancies. It was therefore hypothesized that this locus also plays a role in the development of chondrosarcoma and this locus was investigated at protein, genetic, and epigenetic levels. Loss of p16 protein expression was detected by immunohistochemistry in 12 of 73 central chondrosarcomas and it correlated with increasing histological grade (p = 0.001). Loss of p16 protein expression was not found in 51 enchondromas, which are presumed to be potential precursors of conventional central chondrosarcoma. LOH at 9p21 was found in 15 of 39 chondrosarcomas (38%) but it did not correlate with loss of p16 protein expression. SSCP analysis of p16 did not reveal any mutations in 47 cases. Also, p14 was not the target of LOH, since it gave no aberrant bands on SSCP. To investigate whether an epigenetic mechanism was operating, methylation‐specific PCR was used to look at p16 promotor methylation, which was identified in 5 of 30 tumours. However, this did not correlate with protein expression, or with LOH at 9p21. Cytogenetic data were available in a subset of cases. All tumours that showed chromosome 9 alterations also showed LOH and loss of INK4A/p16 protein expression. It is concluded that although some alterations were found at the DNA level and at the promoter expression level, the lack of correlation between LOH, promotor methylation, and protein expression indicates that a locus other than CDKN2A/p16 must be the target of LOH at 9p21. The correlation between INK4A/p16 protein expression and tumour grade, and the retention of expression in enchondromas, indicates that loss of INK4A/p16 protein expression may be an important event during tumour progression from enchondroma to conventional central chondrosarcoma, and in the progression in grade after recurrence of chondrosarcoma. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Phosphorylated KDR is expressed in the neoplastic and stromal elements of human renal tumours and shuttles from cell membrane to nucleus
Authors: Fox Stephen B , Turley Helen , Cheale Michelle , Blázquez Cristina , Roberts Helen , James Neil , Cook Nathan , Harris Adrian , Gatter Kevin
Published: February 2004

Vascular endothelial growth factor (VEGF)‐A is an important angiogenic factor in establishing the vasculature in renal cell carcinomas (RCCs). Since little is known about VEGF signalling in RCCs, the profile of phosphorylated KDR (pKDR) has been investigated and the intracellular location of the receptor has been examined in the present study. Using two monoclonal antibodies raised against the phosphorylated KDR epitopes (Y1059 and Y1214) known to mediate different VEGF functions, together with a commercial anti‐KDR antibody and immunohistochemistry, the expression of pKDR was investigated in a series of normal (n = 25) and neoplastic kidneys (n = 54; clear cell n = 35; papillary n = 10; oncocytomas n = 8). pKDR was present in many tissue elements of both normal and neoplastic renal tissues, with strong expression in the cell membrane, cytoplasm, and nuclei of normal kidney and tumour cells, as well as endothelial cells in tumours of all histological types. Patterns and intensity were similar using both anti‐pKDR antibodies. There was no significant correlation in clear cell carcinomas between pKDR expression and age (p = 0.57), tumour size (p = 0.2), gender (p = 0.59), grade (p = 0.2) or histological type (p = 0.36). To delineate further the intracellular processing that might account for the cellular distribution, confocal microscopy was also performed. Antibodies to the different phosphorylated epitopes demonstrated different intracellular staining patterns. This study shows that pKDR is present in a wide variety of renal tumours, suggesting that anti‐VEGF therapy might have direct effects on tumour cells. It further suggests that cells traffic pKDR depending on the precise KDR tyrosines that are autophosphorylated in a manner that enables receptor activation to result in different functions. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low‐grade serous tumours
Authors: Sieben Nathalie LG , Macropoulos Patricia , Roemen Guido MJM , Kolkman‐Uljee Sandra M , Jan Fleuren Gert , Houmadi Rifat , Diss Tim , Warren Bretta , Al Adnani Mudher , de Goeij Anton PM , Krausz Thomas ,   The Cancer Genome Project, Flanagan Adrienne M
Published: February 2004

Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS/RAF/MEK/ERK/MAP‐kinase pathway. Activating mutations in BRAF have recently been identified in melanomas, colorectal cancers, and thyroid and ovarian tumours. In the present study, an extensive characterization of BRAF and KRAS mutations has been performed in 264 epithelial and non‐epithelial ovarian neoplasms. The epithelial tumours ranged from adenomas and borderline neoplasms to invasive carcinomas including serous, mucinous, clear cell, and endometrioid lesions. It is shown that BRAF mutations in ovarian tumours occur exclusively in low‐grade serous neoplasms (33 of 91, 36%); these included serous borderline tumours (typical and micropapillary variants), an invasive micropapillary carcinoma and a psammocarcinoma. KRAS mutations were identified in 26 of 91 (29.5%) low‐grade serous tumours, 7 of 49 (12%) high‐grade serous carcinomas, 2 of 6 mucinous adenomas, 22 of 28 mucinous borderline tumours, and 10 of 18 mucinous carcinomas. Of note, two serous borderline tumours were found to harbour both BRAF and KRAS mutations. The finding that at least 60% of serous borderline tumours harbour mutations in two members of the ERK‐MAP‐kinase pathway (BRAF 36%, KRAS 30%) compared with 12% of high‐grade serous carcinomas (BRAF 0%, KRAS 12%) indicates that the majority of serous borderline tumours do not progress to serous carcinomas. Furthermore, no BRAF mutations were detected in the other 173 ovarian tumours in this study. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Comparative multi‐methodological measurement of ERBB2 status in breast cancer
Authors: Ginestier Christophe , Charafe‐Jauffret Emmanuelle , Penault‐Llorca Frédérique , Geneix Jeannine , Adélaïde José , Chaffanet Max , Mozziconacci Marie‐Joëlle , Hassoun Jacques , Viens Patrice , Birnbaum Daniel , Jacquemier Jocelyne
Published: February 2004

The ERBB2 transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target in breast cancer. Accurate determination of ERBB2 status is a prerequisite for the establishment of prognostic significance and for the selection of ERBB2‐overexpressing breast tumours for specific treatment. Unfortunately, there is no complete agreement on how this determination should be performed. This study has compared four methods of assessment of ERBB2 status. Two global, extraction‐based methods using real‐time quantitative PCR on DNA (Q‐PCR) or RNA (RQ‐PCR) and two non‐global, tissue‐based methods, ie fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were used. The 94 breast cancers tested were enriched in cases scoring 2+ using the IHC scoring system currently in use and for which the actual ERBB2 status remains ill defined. To determine the best parameters and reagents for assessment, two protocols for FISH and five anti‐ERBB2 antibodies were used, and both FISH and IHC were carried out on the same tissue microarrays (TMAs). It is shown that the combination of the two tissue‐based methods gives the best results. The use of either PCR‐based method did not improve the results significantly. A new combined IHC score based on the association of two selected anti‐ERBB2 antibodies (HercepTest™ and TAB250) and a dual scale for improved assessment of ERBB2 protein expression, particularly in 2+ cases, are proposed. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Distribution of laminins in the basement membranes of the upper gastrointestinal tract and Barrett's oesophagus
Authors: Dave U , Thursz MR , Ebrahim HY , Burke MM , Townsend ER , Walker MM
Published: February 2004

Barrett's oesophagus predisposes to oesophageal adenocarcinoma. In vitro, laminin, a component of the epithelial basement membrane (BM), is important in regulation of cell differentiation. There is limited information on the distribution of laminin chains in the upper gastrointestinal tract (GIT) and none in Barrett's oesophagus. This study aimed to investigate qualitatively the distribution of laminins in the normal upper GIT mucosa and Barrett's oesophagus in order to understand the role of laminins in metaplasia. Immunoperoxidase staining for laminin chains α1, α2, α3, α5, β1, β2, β3, γ1, and γ2 was performed on frozen endoscopic squamous and Barrett's oesophageal biopsies and surgical resection specimens from squamous oesophagus (in resection specimens for oesophageal cancer), and in oesophageal and gastric biopsies from control subjects. α1 laminin was expressed in the BM of submucosal glands and ducts in squamous oesophagus and Brunner's glands in the duodenum, but not in Barrett's oesophagus or elsewhere in the upper GIT. α2 laminin chain was expressed in a granular distribution in the BM of squamous epithelium. In columnar epithelium, including Barrett's oesophagus, α2 laminin chain was expressed continuously in the BM of glands and deeper pits, but expression was reduced and granular in the surface epithelial BM. β2 laminin was continuous in squamous epithelial BM, but in Barrett's and cardia, gastric body, and duodenum, it was expressed faintly in the surface but continuously in the BM of glands and deeper pits. The constituents of laminin‐5 were continuously expressed in the BM of squamous epithelium, but in the cardia, gastric body, duodenum, and Barrett's, they were expressed only in the BM of surface epithelium, with a sharp decline in the glandular and deeper pit BM. Site‐specific distribution of the α2 and β2 laminin chains may therefore have an important role in Barrett's metaplasia. However, the absence of α1 laminin in Barrett's mucosa suggests that this is unlikely to play an important role in columnar metaplasia. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Vascular endothelial growth factor (VEGF) induces matrix metalloproteinase expression in immortalized chondrocytes
Authors: Pufe Thomas , Harde Viola , Petersen Wolf , Goldring Mary B , Tillmann Bernhard , Mentlein Rolf
Published: February 2004

VEGF (vascular endothelial growth factor), an important angiogenesis factor, appears also to be involved in inflammatory processes. Recent studies have shown that VEGF and its receptors (VEGFR) are expressed on osteoarthritic, but not on normal adult, chondrocytes. To elucidate possible functions of VEGF in osteoarthritic cartilage, the effects of VEGF were studied on immortalized human chondrocytes. Activated matrix metalloproteinase (MMP)‐1, MMP‐3, MMP‐13, tissue inhibitor of metalloproteinases (TIMP)‐1, TIMP‐2, interleukin (IL)‐1β, IL‐6, and tumour necrosis factor‐α (TNF‐α) were measured in culture supernatants by enzyme‐linked immunosorbent assays, nitric oxide with the Griess reagent, and cell proliferation by [3H]thymidine incorporation. VEGFR‐2 mRNA was quantified by real‐time reverse transcription‐polymerase chain reaction and the protein was identified by immuno‐gold electron microscopy. Intracellular signal transduction effects were determined by western blots and electrophoretic mobility shift assays. The chondrocyte cell lines C28/I2, C20/A4, and T/C28a2/a4 expressed functionally active VEGFR‐2. VEGF stimulation induced receptor phosphorylation, activation of the mitogen‐activated protein kinases ERK 1/2, and long‐lasting activation of the transcription factor AP‐1 (activator protein‐1). VEGF increased secreted MMP‐1, MMP‐3, and especially MMP‐13, which could be effectively reduced by an inhibitor of VEGFR‐2 kinase activity. Interestingly, VEGF diminished the expression of TIMP‐1 and especially TIMP‐2. Under hypoxic conditions, as occur in cartilage, the reduction in TIMP levels was even greater. Furthermore, VEGF induced IL‐1β, IL‐6, TNF‐α, and nitric oxide expression to a small extent and stimulated the proliferation of immortalized chondrocytes. These findings indicate that VEGF is an autocrine stimulator of immortalized chondrocytes that mediates mainly destructive processes in osteoarthritis. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Distribution and significance of 14‐3‐3σ, a novel myoepithelial marker, in normal, benign, and malignant breast tissue
Authors: Simpson Peter T , Gale Theodora , Reis‐Filho Jorge S , Jones Chris , Parry Suzanne , Steele Dawn , Cossu Antonio , Budroni Mario , Palmieri Giuseppe , Lakhani Sunil R
Published: February 2004

14‐3‐3σ is a candidate tumour suppressor gene transactivated by p53 in response to DNA damage. In gene expression analysis of normal luminal and myoepithelial cells, 14‐3‐3σ was preferentially expressed by myoepithelial cells. This study has analysed the immunohistochemical distribution and subcellular localization of 14‐3‐3σ in normal breast tissue and in a large series of benign and malignant breast lesions on whole tissue sections and by tissue microarray. Immunohistochemistry demonstrated that 14‐3‐3σ was consistently expressed in the cytoplasmic compartment and occasionally in the nuclei of myoepithelial cells arranged as a continuous layer around normal ducts and lobular units, whereas luminal epithelial, stromal, endothelial, pericytic, lipomatous, and neural cells showed no staining. Myoepithelial cells of benign proliferations and pre‐invasive lesions were consistently positive for 14‐3‐3σ. Strong expression of 14‐3‐3σ was evident in one case of ductal carcinoma in situ (5.5%) and in 105/554 invasive cancers (18.9%). Survival data were available for 452 patients with invasive breast carcinoma. 14‐3‐3σ cytoplasmic subcellular localization was a statistically significant prognostic factor for the whole series of invasive carcinomas, as well as for those positive for oestrogen (ER) or progesterone receptors (PR). This analysis demonstrates the utility of 14‐3‐3σ as a new adjunct antibody for characterization of myoepithelial cells and myoepithelial lesions and it may be a novel prognostic factor for breast cancer patients. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
H1‐Antihistaminika als Allergene? Pharmakophore und Epitope
Authors: Siebert C. D.
Published: February 2004

Beim Vergleich der beschriebenen H1‐Antihistaminika ist offensichtlich, dass Hautreaktionen in verschiedenen Erscheinungsformen die am häufigsten auftretenden allergischen Nebenwirkungen sind. Sie werden von allen hier erwähnten Arzneistoffen verursacht, vermutlich mit Ausnahme von Levocetirizin, haben aber nur sehr selten eine schwere Verlaufsform (wie z.B. das Stevens‐Johnson‐Syndrom bei Ketotifen). Bei etwa der Hälfte aller H1‐Blocker sind ein allergisches Asthma mit Bronchospasmus und damit verbunden Atemprobleme beschrieben worden. Gleiches trifft auch für den anaphylaktischen Schock zu, der durch Organversagen zum Tode führen kann. Quincke‐Ödeme bzw. angioneurotische Ödeme sind bei den Antihistaminika der ersten Generation nicht beschrieben worden, mit Ausnahme des auch strukturell abweichenden Dimetindens. Damit wird deutlich, dass die IgE‐vermittelten Reaktionen vom Soforttyp die größten Risiken darstellen. Die allergische Rhinitis spielt eine untergeordnete Rolle und tritt als einzige allergische Nebenwirkung bei Levocabastin auf. Mit einer Photosensibilität muss vor allem bei den Antihistaminika der ersten Generation und bei Promethazin gerechnet werden. Auffallend ist, dass zu Levocetirizin keine Angaben zu allergischen Reaktionen gefunden wurden, so dass vermutet werden kann, dass die für das racemische Cetirizin beschriebenen Allergien dem weniger wirksamen Enantiomer zugeschrieben werden können. Eine Auffälligkeit gilt es noch für die Typ‐II‐Allergien (Agranulozytose, aplastische Anämie und thrombozytopenische Purpura) zu erwähnen. Sie wurden ausschließlich bei den Dexchlorpheniramin‐ verwandten Substanzen gefunden.

Es konnte exemplarisch gezeigt werden, dass die vergleichende Strukturanalyse ein probates Hilfsmittel zur Bewertung von auftretenden Allergien ist. Wie die Beispiele von Ibuprofen und Disopyramid zeigen, muss das Epitop nicht immer deckungsgleich mit dem Pharmakophor sein, was bedeutet, dass Allergien auf Arzneistoffe unterschiedlicher Indikation chemisch und nicht pharmakologisch klassifiziert werden sollten. Konsequent weiter gedacht bedeutet dies, dass die Gesamtzahl von in der Literatur beschriebenen verschiedenen Arzneistoffallergien deutlich abnimmt, was zugleich hilfreich für eine Systematisierung ist.
The moral case for the estate tax
Authors: Pactor Andrea K.
Published: February 2004

A historical review of philosophers who have considered the role of the wealthy in society is presented and the moral implications of this controversial tax discussed.
Responsible grant making
Authors: Comstock Paul L.
Published: February 2004

Making a responsible grant requires a thoughtful and well‐articulated statement of intent and an implementation process that is developed and followed by the family members. The emphasis should be on fulfilling agreed‐upon objectives in the most effective, efficient, and consensual way, and the process should encourage the emergence of future leadership.
An altruistic reanalysis of the social support hypothesis: The health benefits of giving
Authors: Brown Stephanie L.
Published: February 2004

Can helping others be good for our health and well‐being? This chapter summarizes recent research that offers new evidence in favor of this possibility.
Intergenerational service learning and volunteering
Authors: Butts Donna M.
Published: February 2004

We must move beyond the traditional thinking that older people are our past and younger people are our future. The potential and promise of both these populations must be found if we are to continue to build the legacy of volunteerism and philanthropy that is fundamental to our civil society.
Tracking giving across generations
Authors: Steinberg Richard , Wilhelm Mark
Published: February 2004

The Center on Philanthropy Panel Study (COPPS) reports on the giving and volunteering of more than seventy‐four hundred households in 2001 and 2003 and the households' composition, income, and wealth over the previous thirty years. These data will help researchers and fundraisers understand many aspects of philanthropy not previously understood.
Leadership in emerging family philanthropy
Authors: Lathram Lorna M.
Published: February 2004

As new donors approach philanthropy, many look first to define their work and then to which vehicle they might use to accomplish it. Also, many are making more collaborative efforts to address complex social issues. In this chapter, the former board chair of the Foundation Incubator discusses addressing the needs of this emerging audience.
Differential protein expression assessed by two‐dimensional gel electrophoresis for two wheat varieties grown at four nitrogen levels
Authors: Bahrman Nasser , Le Gouis Jacques , Negroni Luc , Amilhat Laurence , Leroy Philippe , Lainé Anne‐Lyse , Jaminon Odile
Published: February 2004

To limit N‐fertilizer applied on wheat, cultivars that use N more efficiently are needed. Our objective was to investigate differences of nitrogen utilization in varieties by studying qualitative and quantitative proteins expression. Two wheat varieties, ‘Arche’ and ‘Récital’, were grown under controlled conditions at four N levels (0, 2, 8, and 20 mg N/plant/day) with two replicates. The number of tillers/plant, aerial dry weight/plant and total N content were measured after two months. Two‐dimensional gel electrophoresis was also performed on leaf protein extracts. Analyses of variance showed that the N level effect was highly significant for the number of tillers/plant, aerial dry weight and N content. The variety×N level interaction was significant for N content. Analyses of variance on % volume carried out for 524 spots showed a significant variety effect for 55 spots and a significant N treatment effect for 76 spots. Twenty spots showed a significant variety×N treatment interaction. Fourteen proteins were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The possible role of these proteins, eight of which belong to the carbon metabolism, is discussed.
Proteomic analysis of lymph
Authors: Leak Lee V. , Liotta Lance A. , Krutzsch Henry , Jones Michael , Fusaroa Vincent A. , Ross Sally J. , Zhao Yingming , Petricoin Emanuel F.
Published: February 2004

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS), two‐dimensional gel electrophoresis (2‐D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI‐TOF‐MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2‐D PAGE analysis. MS analysis of a large number of protein spots from 2‐D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2‐D PAGE gels of lymph included, fibrinogen α‐ and β‐chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A‐1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor‐1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.
Ovarian cancer detection by logical analysis of proteomic data
Authors: Alexe Gabriela , Alexe Sorin , Liotta Lance A. , Petricoin Emanuel , Reiss Michael , Hammer Peter L.
Published: February 2004

A new type of efficient and accurate proteomic ovarian cancer diagnosis systems is proposed. The system is developed using the combinatorics and optimization‐based methodology of logical analysis of data (LAD) to the Ovarian Dataset 8‐7‐02 (http://clinicalproteomics.steem.com), which updates the one used by Petricoin et al. in The Lancet 2002, 359, 572–577. This mass spectroscopy‐generated dataset contains expression profiles of 15 154 peptides defined by their mass/charge ratios (m/z) in serum of 162 ovarian cancer and 91 control cases. Several fully reproducible models using only 7–9 of the 15 154 peptides were constructed, and shown in multiple cross‐validation tests (k‐folding and leave‐one‐out) to provide sensitivities and specificities of up to 100%. A special diagnostic system for stage I ovarian cancer patients is shown to have similarly high accuracy. Other results: (i) expressions of peptides with relatively low m/z values in the dataset are shown to be better at distinguishing ovarian cancer cases from controls than those with higher m/z values; (ii) two large groups of patients with a high degree of similarities among their formal (mathematical) profiles are detected; (iii) several peptides with a blocking or promoting effect on ovarian cancer are identified.
Effect of collagen substrates on proteomic modulation of breast cancer cells
Authors: Fontana Simona , Pucci‐Minafra Ida , Becchi Michel , Freyria Anne‐Marie , Minafra Salvatore
Published: February 2004

We have previously described the occurrence, in breast and colon cancer extra‐cellular matrix, of an oncofoetal form of collagen, OF/LB, able to induce an increase in cell proliferation and motility in the breast cancer cell line 8701‐BC. It also caused an increased amount of type V collagen which appears to exert an anti‐proliferative effect on the same cells. The aim of the present study was to investigate, at the proteomic level, the effect of OF/LB and type V collagens used as substrates for neoplastic cell growth. Due to the complexity of a whole proteomic profile, a subset of significant protein classes was used to assess variations in protein expression levels. For this study we adopted a multivariate statistical procedure that allows a global view of the variations induced by different growth conditions, when several variables have to be analyzed simultaneously. The results of this research indicate that in response to different growth substrates, chaperons and heat shock proteins contributed most to the dissimilarity in levels of expression of the selected protein spots. Moreover, we observed that different isoforms of the same protein showed independent levels of expression from one another in relation to the different collagen treatments.
Identification and characterization of phosphorylated proteins in the human pituitary
Authors: Giorgianni Francesco , Beranova‐Giorgianni Sarka , Desiderio Dominic M.
Published: February 2004

Post‐translational modifications of proteins from the human pituitary gland play an important role in the regulation of different body functions. We report on the application of a liquid chromatography‐tandem mass spectrometry (MS/MS) based approach to detect and characterize phosphorylated proteins in a whole human pituitary digest. By combining an immobilized metal affinity column‐based enrichment method with MS/MS conditions that favor the neutral loss of phosphoric acid from a phosphorylated precursor ion, we identified several previously undescribed phosphorylated peptides. The identified peptides were matched to the sequences of six pituitary proteins: the human growth hormone, chromogranin A, secretogranin I, 60S ribosomal protein P1 and/or P2, DnaJ homolog subfamily C member 5, and galanin. The phosphorylation sites of these important regulatory proteins were determined by MS/MS and MS3 analysis.
Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal‐to‐background ratio and improved baseline resolution
Authors: Lamanda Andreas , Zahn Alain , Röder Daniel , Langen Hanno
Published: February 2004

In proteomics the ability to visualize proteins from electropherograms is essential. Here a new protocol for staining and destaining gels treated with Ruthenium II tris (bathophenantroline disulfonate) is presented. The method is compared with the silver‐staining procedure of Swain and Ross, the Ruthenium II tris (bathophenantroline disulfonate) stain described by Rabilloud (Rabilloud T., Strub, S. M. Luche, S., Girardet, S. L. et al., Proteomics 2001, 1, 699–704) and the SYPRO Ruby gel stain. The method offers a better signal‐to‐background ratio with improved baseline resolution for both sodium dodecyl sulfate‐polyacrylamide gels and two‐dimensional gels.
About thiol derivatization and resolution of basic proteins in two‐dimensional electrophoresis
Authors: Luche Sylvie , Diemer Hélène , Tastet Chistophe , Chevallet Mireille , Van Dorsselaer Alain , Leize‐Wagner Emmanuelle , Rabilloud Thierry
Published: February 2004

The influence of thiol blocking on the resolution of basic proteins by two‐dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in‐gel rehydration on wide gradients (e.g. 3–10 and 4–12), but anodic cup‐loading was still required for basic gradients (e.g. 6–12 or 8–12). These results demonstrate that thiol‐related problems are not solely responsible for streaking of basic proteins on two‐dimensional gels.
Evaluation of algorithms for protein identification from sequence databases using mass spectrometry data
Authors: Chamrad Daniel C. , Körting Gerhard , Stühler Kai , Meyer Helmut E. , Klose Joachim , Blüggel Martin
Published: February 2004

In this work, the commonly used algorithms for mass spectrometry based protein identification, Mascot, MS‐Fit, ProFound and SEQUEST, were studied in respect to the selectivity and sensitivity of their searches. The influence of various search parameters were also investigated. Approximately 6600 searches were performed using different search engines with several search parameters to establish a statistical basis. The applied mass spectrometric data set was chosen from a current proteome study. The huge amount of data could only be handled with computational assistance. We present a software solution for fully automated triggering of several peptide mass fingerprinting (PMF) and peptide fragmentation fingerprinting (PFF) algorithms. The development of this high‐throughput method made an intensive evaluation based on data acquired in a typical proteome project possible. Previous evaluations of PMF and PFF algorithms were mainly based on simulations.
Proteomic analysis of κ‐casein micro‐heterogeneity
Authors: Holland John W. , Deeth Hilton C. , Alewood Paul F.
Published: February 2004

The proteome of bovine milk is dominated by just six gene products that constitute approximately 95% of milk protein. Nonetheless, over 150 protein spots can be readily detected following two‐dimensional electrophoresis of whole milk. Many of these represent isoforms of the major gene products produced through extensive post‐translational modification. Peptide mass fingerprinting of in‐gel tryptic digests (using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) in reflectron mode with α‐cyano‐4‐hydroxycinnamic acid as the matrix) identified 10 forms of κ‐casein with isoelectric point (pI) values from 4.47 to 5.81, but could not distinguish between them. MALDI‐TOF MS in linear mode, using sinapinic acid as the matrix, revealed a large tryptic peptide (mass > 5990 Da) derived from the C‐terminus that contained all the known sites of genetic variance, phosphorylation and glycosylation. Two genetic variants present as singly or doubly phosphorylated forms could be distinguished using mass data alone. Glycoforms containing a single acidic tetrasaccharide were also identified. The differences in electrophoretic mobility of these isoforms were consistent with the addition of the acidic groups. While more extensively glycosylated forms were also observed, substantial loss of N‐acetylneuraminic acid from the glycosyl group was evident in the MALDI spectra such that ions corresponding to the intact glycopeptide were not observed and assignment of the glycoforms was not possible. However, by analysing the pI shifts observed on the two‐dimensional gels in conjunction with the MS data, the number of N‐acetylneuraminic acid residues, and hence the glycoforms present, could be determined.
Construction of a two‐dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow‐2 cell suspension culture
Authors: Laukens Kris , Deckers Peter , Esmans Eddy , Van Onckelen Harry , Witters Erwin
Published: February 2004

Using two‐dimensional gel electrophoresis (2‐DE) and electrospray‐tandem mass spectrometry (ESI‐MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow‐2 (tobacco BY‐2). The BY‐2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2‐DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi‐automated liquid chromatography‐ESI‐quadrupole time of flight‐tandem MS (LC‐ESI‐QTOF‐MS‐MS) setup and cross‐species matching. These data were integrated in a database, which can be accessed at http://tby2‐www.uia.ac.be/tby2/. On the on‐line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on‐line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY‐2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY‐2 proteomics in the future.
Two‐dimensional gel electrophoresis pattern (pH 6–11) and identification of water‐soluble barley seed and malt proteins by mass spectrometry
Authors: Bak‐Jensen Kristian Sass , Laugesen Sabrina , Roepstorff Peter , Svensson Birte
Published: February 2004

A protocol was established for two‐dimensional gel electrophoresis (2‐DE) of barley seed and malt proteins in the pH range of 6–11. Proteins extracted from flour in a low‐salt buffer were focused after cup‐loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS‐PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty‐seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2‐D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in‐gel digestion by trypsin. In addition, the N‐terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2‐D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting.
Comparative proteome analysis of thalamus in MK‐801‐treated rats
Authors: Paulson Linda , Martin Peter , Nilsson Carol L. , Ljung Elisabeth , Westman‐Brinkmalm Ann , Blennow Kaj , Davidsson Pia
Published: February 2004

Two‐dimensional gel‐electrophoresis in combination with mass spectrometry is a powerful approach to compare protein expression in brain tissues. Using this proteomic approach, and based on the hypothesis that schizophrenia involves hypoglutamergic brain function, alterations in protein levels in the thalamus of rats treated with the N‐methyl‐D‐aspartate (NMDA) receptor antagonist [+]‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo‐[a,d]‐cycloheptene‐5,10‐iminehydrogenmaleate (MK‐801), as compared to saline‐treated animals, were assessed in an unbiased fashion. The rats were divided into two groups; group 1 (short‐term treated) and group 2 (long‐term treated). In group 1, the levels of seven proteins were increased and four proteins reduced. In group 2, the levels of six proteins were reduced. Several of the altered proteins (heat shock proteins 60 and 72, albumin, dihydropyrimidinase related protein‐2, aldolase c, and malate dehydrogenase) have previously been connected to schizophrenia. Alterations of other proteins (dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex E2, guanine deaminase, α‐enolase, aconitase, ATP‐synthase and α‐internexin), have not, to the best of our knowledge, earlier been implicated in schizophrenia pathology. Our results show the high potential of using proteomic methods for the validation of animal models of schizophrenia and to identify new proteins involved in the pathophysiological mechanisms of schizophrenia.
Comparative proteomic analysis of de‐N‐glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status
Authors: Comunale Mary Ann , Mattu Taj S. , Lowman Melissa A. , Evans Alison A. , London W. Thomas , Semmes O. John , Ward Michael , Drake Richard , Romano Patrick R. , Steel Laura F. , Block Timothy M. , Mehta Anand
Published: February 2004

Analysis of the polypeptide profile in tissues, cells, and sera by high‐resolution two‐dimensional (2‐D) gel electrophoresis offers promise in the identification of biomarkers that correlate with disease. However, sera contain many polypeptides bearing N‐linked glycosylation that can complicate interpretation. Therefore, we tested the possibility that de‐N‐glycosylation of the polypeptides present in human serum would result in a simplification of serum proteome profiles. Briefly, polypeptides present in human serum were left untreated or subjected to de‐N‐glycosylation by incubation with PNGase F and resolved by high‐resolution 2‐D gel electrophoresis. De‐N‐glycosylation reduced the number of glycoform variants, enhanced the resolution of many polypeptides and allowed other polypeptides to become visible. As an initial test of concept, clinically relevant serum samples from individuals with or without diagnosis of hepatocellular carcinoma were compared. Several polypeptides, apparent only after de‐N‐glycosylation, were shown to correlate with disease. Although the results are preliminary and the identities of all the putative biomarkers not yet known, the data suggest that de‐N‐glycosylation offers a method to enhance the resolution of serum polypeptide profiles and has value in comparative proteomic studies.
Protein profiling and identification of modulators regulated by the E7 oncogene in the C33A cell line by proteomics and genomics
Authors: Lee Kyung‐Ae , Shim Jung‐Hyun , Kho Chang Won , Park Sung Goo , Park Byoung Chul , Kim Jae‐Wha , Lim Jong‐Seok , Choe Yong‐Kyung , Paik Sang‐Gi , Yoon Do‐Young
Published: February 2004

Human papillomaviruses (HPVs) have been recognized as the primary cause of cervical cancer. HPV 16 E7 binds to tumor suppressor retinoblastoma protein, and interferes with its function, causing release of the transcription factor E2F, which influences expression of cell cycle‐related genes. This study was performed to identify the genes and proteins modulated by the HPV E7 oncogene. An HPV‐negative cervical cancer cell line (C33A) was prepared to establish a stable cell line expressing E7. In order to analyze the target molecules modulated by E7 expression, we used two approaches: matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) and DNA microarrays. Forty‐seven spots were identified in C33A/E7 by two‐dimensional electrophoresis and MALDI/TOF MS. Protein disulfide isomerase A3, integrase interactor 1 protein, growth inhibitory protein, glutathione S‐transferase P, and vav proto‐oncogene were down‐regulated, whereas heat shock 60 kDa protein 1, Ku70 binding protein, alpha enolase, 26S proteasome subunit were up‐regulated. A genomic approach using a microarray kit showed that IL‐12Rβ1, cytochrome c, and tumor necrosis factor receptor II were induced by the E7 oncogene. These results suggest that E7 can evade immune surveillance by suppressing or inducing these cell signaling factors, cell cycle regulators, and chaperones.
A map of WW domain family interactions
Authors: Hu Hai , Columbus John , Zhang Yi , Wu Dongying , Lian Lubing , Yang Song , Goodwin Jennifer , Luczak Christine , Carter Mark , Chen Lin , James Michael , Davis Roger , Sudol Marius , Rodwell John , Herrero Juan J.
Published: February 2004

WW domains are protein modules that bind proline‐rich ligands. WW domain‐ligand complexes are of importance as they have been implicated in several human diseases such as muscular dystrophy, cancer, hypertension, Alzheimer's, and Huntington's diseases. We report the results of a protein array aimed at mapping all the human WW domain protein‐protein interactions. Our biochemical approach integrates parallel synthesis of peptides, protein expression, and high‐throughput screening methodology combined with tools of bioinformatics. The results suggest that the majority of the bioinformatically predicted WW peptide ligands and most WW domains are functional, and that only about 10% of the measured domain‐ligand interactions are positive. The analysis of the WW domain protein arrays also underscores the importance of the amino acid residues surrounding the WW ligand core motifs for specific binding to WW domains. In addition, the methodology presented here allows for the rapid elucidation of WW domain‐ligand interactions with multiple applications including prediction of exact WW ligand binding sites, which can be applied to the mapping of other protein signaling domain families. Such information can be applied to the generation of protein interaction networks and identification of potential drug targets. To our knowledge, this report describes the first protein‐protein interaction map of a domain in the human proteome.
Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae)
Authors: Wang Sheng‐Bing , Hu Qiang , Sommerfeld Milton , Chen Feng
Published: February 2004

The green microalga Haematococcus pluvialis can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, and undergo profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stresses. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) coupled with peptide mass fingerprinting (PMF) and sequence‐database analysis. In total, 163 protein bands were analyzed, which resulted in positive identification of 81 protein orthologues. The highly complex and dynamic composition of CWPs is manifested by the fact that the majority of identified CWPs are differentially expressed at specific stages of the cell cycle along with a number of common wall‐associated ‘housekeeping’ proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation, in contrast to earlier observations that cellulose was exclusively present in the secondary wall of the organism. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation and/or assisting in cell expansion. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and the specific protein markers obtained from this study provide a framework for future characterization of the expression and physiological functions of the proteins involved in the biogenesis and modifications in the cell wall of Haematococcus and related organisms.
Proteomic analysis of hyperoxia‐induced responses in the human choriocarcinoma cell line JEG‐3
Authors: Vorum Henrik , Østergaard Morten , Hensechke Philip , Enghild Jan J. , Riazati Mahtab , Rice Gregory E.
Published: February 2004

Living cells exposed to changes in the surrounding oxygen tension, have the ability to adapt to the new environment through the regulatory effect of intracellular mediators. In an effort to identify important proteins that may be involved in the hyperoxic response, we performed proteomic analysis on the human choriocarcinoma cell line JEG‐3, incubated under high oxygen tension (carbogen, 95% O2/5% CO2) or air (21% oxygen/5% CO2). We identified 13 protein spots that were significantly down‐regulated (p < 0.05) in JEG‐3 cells incubated under hyperoxic conditions compared to standard conditions. Ten of these spots were positively identified by matrix‐assisted laser desorption/ionization time of flight‐mass spectrometry as nine different proteins: Villin 2, tublin β, profilin I, glyceraldehyde‐3‐phosphate dehydrogenase, phosphoglycerate mutase, peroxiredoxin 1, neuroplypeptide h3, poly(rC)‐binding protein 1 and cyclophilin A. These proteins have been implicated in regulating cytoskeletal structure, glycolysis, redox status, signal transduction, transcription and protein folding. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation and motility.