bluuebunny/crossref_metadata_2025 huggingface dataset:
Clustering of deletions on chromosome 13 in benign and low‐malignant lipomatous tumors
Authors: Dahlén Anna , Debiec‐Rychter Maria , Pedeutour Florence , Domanski Henryk A. , Höglund Mattias , Bauer Henrik C. F. , Rydholm Anders , Sciot Raf , Mandahl Nils , Mertens Fredrik
Published: December 2002

Deletions and structural rearrangements of the long arm of chromosome 13 are frequently observed in benign and low‐malignant lipomatous tumors, but nothing is known about their molecular genetic consequences. We assessed the karyotypes of 40 new and 22 previously published cases (35 ordinary lipomas, 15 spindle cell/pleomorphic lipomas, 2 myxolipomas, 1 angiomyxolipoma and 9 atypical lipomatous tumors) with chromosome 13‐abnormalities, and found bands 13q12–22 to be frequently affected. Twenty‐seven cases with structural abnormalities within this region were selected for breakpoint and deletion mapping by metaphase fluorescencein situhybridization (FISH), using a set of 20 probes. Deletions were found in 23 of 27 cases. The remaining 4 cases had seemingly balanced rearrangements. The breakpoints were scattered but clustered to band 13q14, and in all cases with unbalanced abnormalities, a limited region within band 13q14 was partially or completely deleted. A deletion within band 13q14 was found together with a breakpoint on the other homologue in 5 cases, 4 of which could be tested further with regard to the status of the retinoblastoma (RB1)‐gene. In all 4 cases, only 1 copy of the gene was deleted. In addition to the breaks and deletions in the vicinity of theRB1‐locus, several other regions of 13q were recurrently affected,e.g., in the vicinity of the hereditary breast cancer (BRCA2; 13q12)‐ and lipoma HMGIC fusion partner (LHFP; 13q13)‐ genes. Our findings strongly indicate that deletion of a limited region (∼2.5 Mbp) within 13q14, distal to theRB1‐locus, is of importance in the development of a subset of lipomatous tumors. © 2002 Wiley‐Liss, Inc.
Hereditary nonpolyposis colorectal cancer: Frequent occurrence of large genomic deletions in MSH2 and MLH1 genes
Authors: Wang Yaping , Friedl Waltraut , Lamberti Christof , Jungck Matthias , Mathiak Micaela , Pagenstecher Constanze , Propping Peter , Mangold Elisabeth
Published: December 2002

Hereditary nonpolyposis colorectal cancer (HNPCC) is often caused by a deficiency in DNA mismatch repair. By using conventional methods of mutation analysis, point mutations in the DNA mismatch repair genes MSH2 and MLH1 have been detected in up to 64% of patients suspected of HNPCC. However, large genomic deletions cannot be detected by these methods. In our study, we applied a semiquantitative multiplex PCR to detect the proportion of large deletions in patients meeting the Bethesda criteria whose tumours exhibited microsatellite instability (MSI). Of 368 unrelated patients, 180 exhibited MSI. In these patients, 68 disease‐causing point mutations (38%) had previously been detected in the MSH2 and MLH1 genes by SSCP, heteroduplex analysis or DHPLC followed by direct sequencing. The remaining 112 patients (including 24 patients with rare missense or other unclarified variants) were examined for large deletions. We identified deletions in 19 patients (10.6%); 11/19 (58%) deletions were located in MSH2 and 8/19 (42%) in MLH1, respectively. The size of deletions ranged from 1 exon to a deletion of a whole gene. Five breakpoints of deletions were sequenced; Alu‐repetitive elements were involved in all of them. In patients meeting the Amsterdam criteria the proportion of large deletions was 12.6%. A similar proportion of deletions was found in the group of patients with a positive family history for colorectal cancer and MSI tumours, not meeting the Amsterdam criteria. The results of our study suggest that large genomic deletions in both MSH2 and MLH1 genes play a considerable role in the pathogenesis of HNPCC and should be part of the routine HNPCC mutation detection protocols. © 2002 Wiley‐Liss, Inc.
Single nucleotide polymorphisms in the XPG gene: Determination of role in DNA repair and breast cancer risk
Authors: Kumar Rajiv , Höglund Lotta , Zhao Chunyan , Försti Asta , Snellman Erna , Hemminki Kari
Published: December 2002

In this study we determined the effect of single nucleotide polymorphisms in the XPG gene on DNA repair and breast cancer susceptibility. Ninety individuals, with previously studied DNA repair rate at 24 hr of 2 types of UV‐specific cyclobutane pyrimidines dimers (CPDs) in skin were genotyped for XPG polymorphism at codon 1104 (exon 15 G>C; Asp > His). The repair rate of TT=C dimer was similar in both wild‐type GG homozygotes and GC heterozygotes, whereas, for TT=T, dimer repair was non‐significantly (Student's t‐test, p = 0.34) lower in GC heterozygotes than wild‐type GG homozygotes. Genotyping of 220 breast cancer cases and 308 controls for the same single nucleotide polymorphism in exon 15 of the XPG gene exhibited marginally significant increased frequency of the variant allele (χ2 3.84, p = 0.05; OR 1.33, 95% CI 1.0–1.8) in cases (C‐allele 0.29) compared to controls (C‐allele 0.24). Combined heterozygote and variant homozygote genotype frequency was also higher in cases than controls (χ2 4.79, p = 0.03; OR 1.50, 95%CI 1.04–2.16). © 2002 Wiley‐Liss, Inc.
Insulin‐like growth factor binding protein‐3 is overexpressed in endothelial cells of mouse breast tumor vessels
Authors: Schmid Michael C. , Bisoffi Marco , Wetterwald Antoinette , Gautschi Elsbeth , Thalmann George N. , Mitola Stefania , Bussolino Federico , Cecchini Marco G.
Published: December 2002

Angiogenesis is a key process in a variety of human diseases, including cancer. The ability to target selectively the tumor vasculature is potentially useful for the diagnosis and treatment of cancer. Still, little information is available regarding markers that are restricted to the ECs of tumor vessels. cDNA array technology allows simultaneous analysis of relative expression levels of a broad spectrum of genes in 2 related cell populations. We used this technology with the aim of identifying markers specific for TECs. TECs were isolated by CD31‐mediated immunomagnetic separation from tumors induced by s.c. injection of NF9006 breast carcinoma cells into syngeneic mice. NECs were isolated from lactating mammary glands. The endothelial nature of isolated cells was confirmed by RT‐PCR using CD31‐specific primers and by uptake of DiI‐Ac‐LDL. Macrophage contamination in the EC isolations could be reasonably ruled out by assessing the expression of the macrophage marker c‐fms. 32P‐labeled cDNA probes generated by reverse transcription from total RNA were hybridized to mouse‐specific gene arrays. Several genes consistently showed differential expression between TECs and NECs. However, expression of only 1 of these genes, IGFBP‐3, was restricted exclusively to ECs. Semiquantitative RT‐PCR revealed 22‐ to 33‐fold differential expression of IGFBP‐3 in the TEC fraction. IGFBP‐3 was overexpressed by a factor of 5 in an additional mouse model of breast carcinoma induced by 4T1.2 tumor cells. These results indicate that IGFBP‐3 is a potential novel marker of angiogenesis. Elucidation of its role in tumor neovascularization may open the possibility of IGFBP‐3 as a therapeutic target for antiangiogenesis. © 2002 Wiley‐Liss, Inc.
Abortions and breast cancer: Record‐based case‐control study
Authors: Erlandsson Gunnar , Montgomery Scott M. , Cnattingius Sven , Ekbom Anders
Published: December 2002

It has been suggested that abortions leave the breast epithelium in a proliferative state with an increased susceptibility to carcinogenesis. Results from previous studies of induced or spontaneous abortions and risk of subsequent breast cancer are contradictory, probably due to methodological considerations. We investigated the relationship between abortions and subsequent breast cancer risk in a case‐control study using prospectively recorded exposure information. The study population comprised women recorded in the population‐based Swedish Medical Birth Register between 1973–91. Cases were defined by linkage of the birth register to the Swedish Cancer Register and controls were randomly selected from the birth register. From the subjects' antenatal care records we abstracted prospectively collected information on induced and spontaneous abortions, as well as a number of potential confounding factors. Relative risk of breast cancer was estimated by odds ratios (OR) with 95% confidence intervals (95% CI). A reduced risk of breast cancer was observed for women with a history of at least 1 compared to no abortions (adjusted OR = 0.84, 95% CI = 0.72–0.99). The adjusted OR decreases step‐wise with number of abortions to 0.59 (95% CI = 0.34–1.03) for 3 or more compared to no abortions. The patterns are similar for induced and spontaneous abortions. In conclusion, neither a history of induced nor spontaneous abortions is associated with an increased risk of breast cancer. Our data suggest a protective effect of pregnancies regardless of outcome. © 2002 Wiley‐Liss, Inc.
Direct interaction with and activation of p53 by SMAR1 retards cell‐cycle progression at G2/M phase and delays tumor growth in mice
Authors: Kaul Ruchika , Mukherjee Sujoy , Ahmed Farid , Bhat Manoj Kumar , Chhipa Rishiraj , Galande Sanjeev , Chattopadhyay Samit
Published: December 2002

The tumor‐suppressor p53 is a multifunctional protein mainly responsible for maintaining genomic integrity. p53 induces its tumor‐suppressor activity by either causing cell‐cycle arrest (G1/S or G2/M) or inducing cells to undergo apoptosis. This function of wild‐type p53 as “guardian of the genome” is presumably achieved by forming molecular complexes with different DNA targets as well as by interacting with a number of cellular proteins, e.g., Mdm2, Gadd45, p21, 14‐3‐3σ, Bax and Apaf‐1. Upon activation, p53 activates p21, which in turn controls the cell cycle by regulating G1 or G2 checkpoints. Here, we report SMAR1 as one such p53‐interacting protein that is involved in delaying tumor progression in vivo as well as in regulating the cell cycle. SMAR1 is a newly identified MARBP involved in chromatin‐mediated gene regulation. The SMAR1 gene encodes at least 2 alternatively spliced variants: SMAR1L (the full‐length form) and SMAR1S (the shorter form). We report that expression of SMAR1S, but not of SMAR1L, mRNA was decreased in most of the human cell lines examined, suggesting selective silencing of SMAR1S. Overexpression of SMAR1S in mouse melanoma cells (B16F1) and their subsequent injection in C57BL/6 mice delays tumor growth. Exogenous SMAR1S causes significant retardation of B16F1 cells in the G2/M phase of the cell cycle compared to SMAR1L. SMAR1S activates p53‐mediated reporter gene expression in mouse melanoma cells, breast cancer cells (MCF‐7) and p53 null cells (K562), followed by activation of its downstream effector, p21. We further demonstrate that SMAR1 physically interacts and colocalizes with p53. These data together suggest that SMAR1 is the only known MARBP that delays tumor progression via direct activation and interaction with tumor‐suppressor p53. © 2002 Wiley‐Liss, Inc.
Analysis of the human APC mutation spectrum in a saccharomyces cerevisiae strain with a mismatch repair defect
Authors: Otsuka Kazunori , Suzuki Takao , Shibata Hiroyuki , Kato Satoshi , Sakayori Masato , Shimodaira Hideki , Kanamaru Ryunosuke , Ishioka Chikashi
Published: December 2002

Somatic APC mutations in colorectal tumors with an RER phenotype reflect excessive frameshift mutations, especially in simple repetition tracts within the coding sequence. Because this type of mutation is characteristic of cells with a deficient DNA MMR system, the APC mutation signature of RER tumors may be attributable to a defect in the MMR system. However, there is little experimental evidence to prove that the spectrum of mutations and the APC gene distribution are directly influenced by MMR system defects. We therefore examined the mutation spectrum of the MCR of the APC gene after transfection into both MMR‐proficient and MMR‐deficient yeast strains and compared it with a previously reported human APC mutation database. Small insertions or deletions in mono‐ or dinucleotide repeats were more common in the MMR‐deficient than in the MMR‐proficient strain (91.2% vs. 38.1%, Fisher's exact test p < 0.0001). Furthermore, the 2 mutation hot spots, 4385–4394(AG)5 and 4661–4666(A)6, found in the yeast system corresponded with those in human tumors. Combining our data with those from human tumors, there appears to be hypermutable mutations in specific simple repetitive sequences within the MCR, which are more prevalent in MMR‐deficient cells and RER tumors than in MMR‐proficient cells and non‐RER tumors. We therefore consider that the differences in the spectra of RER and non‐RER tumors are attributable at least in part to the MMR system of the host cells. © 2002 Wiley‐Liss, Inc.
Tissue microarray‐based analysis shows phospho‐β‐catenin expression in malignant melanoma is associated with poor outcome
Authors: Kielhorn Eric , Provost Elayne , Olsen Drew , D'Aquila Thomas G. , Smith Bradley L. , Camp Robert L. , Rimm David L.
Published: December 2002

Beyond depth of invasion, there are very few prognostic markers to predict outcome in melanoma. It has been shown recently that the β‐catenin oncogene is mutated or shows altered subcellular localization suggesting that activation of β‐catenin mediated signaling plays a role in oncogenesis. We hypothesize that assessment of activated β‐catenin, as detected by a phospho‐specific antibody, may be useful to predict outcome in melanoma. We use immuno‐histochemical analysis of β‐catenin and phospho‐β‐catenin, first to verify the specificity of the phospho‐β‐catenin antibody and then to assay expression in a tissue microarray‐based study. The subcellular localization of β‐catenin is membranous in some cases and cytoplasmic and nuclear in others. We validate the specificity of a ser33/37/thr41 phospho‐β‐catenin antibody in transfected cells and show that the expression is almost exclusively localized to the nucleus in both cultured cells and human tissue. Evaluation of both total and phospho‐β‐catenin antibodies showed that cytoplasmic/nuclear staining was more common in primary lesions, whereas nuclear phospho‐β‐catenin was more common in metastatic lesions. High levels of nuclear phospho‐β‐catenin are associated with significantly worse overall survival (51% vs. 25% overall survival at 5 years, p = 0.046). These results suggest that phospho‐specific antibodies to β‐catenin define a unique subset of cases and that monitoring of phospho‐β‐catenin expression may be useful for assessing prognosis in malignant melanoma. © 2002 Wiley‐Liss, Inc.
Changing epidemiology of nasopharyngeal carcinoma in Hong Kong over a 20‐year period (1980–99): An encouraging reduction in both incidence and mortality
Authors: Lee Anne W.M. , Foo William , Mang Oscar , Sze W.M. , Chappell Rick , Lau W.H. , Ko W.M.
Published: December 2002

Epidemiological data from the Hong Kong Cancer Registry for the period 1980–99 were analyzed. Altogether 21,768 new cases of nasopharyngeal carcinoma (NPC) and 8,664 related deaths were registered. In both genders, the peak incidence occurred in the 50–59 years age group, and this age distribution pattern remained similar throughout. The age‐standardized incidence rate steadily decreased from 28.5 in 1980–84 to 20.2 in 1995–99 per 100,000 males, and from 11.2–7.8 per 100,000 females, resulting in a total decrease of 29% for males and 30% for females over this 20‐year period. The magnitude of total decrease in NPC mortality amounted to 43% and 50%, respectively, as the age‐standardized mortality rate steadily decreased from 13.7 in 1980–84 to 7.8 in 1995–99 per 100,000 males, and from 4.5–2.2 per 100,000 females. The age‐standardized mortality/incidence ratio also decreased from the peak of 0.48 in 1980–84 to 0.39 in 1995–99 for males, and from 0.40–0.29 for females. Females had significantly lower age‐standardized incidence (male/female ratio 2.5–2.6, p < 0.01) and mortality (male/female ratio 3.0–3.5, p< 0.01) throughout the whole period. Furthermore, females had consistently lower mortality/incidence ratio: 0.29 vs. 0.39 in 1995–99. These data are highly suggestive of significant improvement in prevention and control of NPC in Hong Kong. Closer scrutiny of the differences in intrinsic and extrinsic factors between the genders might help to show important factors affecting oncogenesis and prognosis. Possible ways for further reduction of incidence and mortality are discussed. © 2002 Wiley‐Liss, Inc.
Development of an in vitro miniature model to simulate immunoadsorption in patients with systemic lupus erythematosus
Authors: Avenhaus Birgit , Avenhaus Wolfgang , Schneider Matthias , Domschke Wolfram , Gaubitz Markus
Published: December 2002

In SLE, immunoadsorption is used as an adjuvant therapy; however, adsorption profiles and binding mechanisms have not yet been completely investigated. Using a minicolumn filled with the sorbent IMPH with or without the ligand phenylalanine, we developed a model simulating clinical conditions in a reduced scale with a constant ratio of plasma to column volume and a constant plasma flow at room temperature. By desorbing the column, the adsorption efficacy for different antibodies could be measured directly. We demonstrate that the adsorption rate can be increased by a low plasma flow and by covering the column surface. Double perfusion of the same column did not increase the amount of adsorbed antibodies. We further demonstrate that the carrier material without a ligand is unable to bind antibodies or protein. In the IMPH sorbent anti‐dsDNA antibodies were significantly better adsorbed than total IgG or total protein. After a single perfusion of 21 samples, we estimated a mean anti‐dsDNA antibody adsorption rate of 22.5% (±13.6). A group of ten responders with a medium adsorption rate of 35.4% (±6.5) clearly differed from a second group of eleven nonresponders (10.9% ± 4.2). Anti‐cardiolipin antibodies (ACA) were adsorbed in a wide range (IgG type, 2.5–52.7%, IgM type, 1.1–37.8%) while anti‐Ro (SSA) antibody adsorption was negligible. This in vitro minimodel provides a precise simulation of therapeutic immunoadsorption and helps to analyze the binding characteristics of the sorbent IMPH and shows its effectiveness in several antibody subsets of different patients. J. Clin. Apheresis 17:183–189, 2002. © 2002 Wiley‐Liss, Inc.
Direct adsorption of low‐density lipoprotein and lipoprotein(a) from whole blood: Results of the first clinical long‐term multicenter study using DALI apheresis
Authors: Bosch T. , Lennertz A. , Schenzle D. , Dräger J. ,   for the DALI Study Group
Published: December 2002

Direct adsorption of lipoproteins (DALI) is the first low‐density lipoprotein (LDL)‐apheresis technique by which atherogenic LDL and lipoprotein(a) (Lp(a)) can be selectively removed from whole blood without plasma separation. The present study was performed to evaluate the efficacy, selectivity and safety of long‐term DALI apheresis. Sixty‐three hypercholesterolemic coronary patients were treated by weekly DALI sessions. Initial LDL‐cholesterol (C) plasma levels averaged 238 ± 87 mg/dl (range 130–681 mg/dl). On average, 34 sessions (1–45) were performed processing 1.5 patient blood volumes. The primary aim was to acutely reduce LDL‐C by ≥60% per session. To this end, three different adsorber sizes could be employed, i.e., DALI 500, 750, and 1,000, which were used in 4, 73, and 23% of the 2,156 sessions, respectively. On average, 7,387 ml of blood were processed in 116 min per session. This resulted in the following mean acute changes: LDL‐C 198 → 63 mg/dl (−69%), Lp(a) 86 → 32 mg/dl (−64%), triglycerides 185 → 136 mg/dl (−27%). HDL‐C (−11%) and fibrinogen (−15%) were not significantly influenced. The mean long‐term reduction of LDL‐C was 42% compared to baseline while HDL‐C slightly increased in the long run (+4%). The selectivity of LDL removal was good as recoveries of albumin, immunoglobulins, and other proteins exceeded 85%. Ninety‐five percent of 2,156 sessions were completely uneventful. The most frequent adverse effects were hypotension (1.2% of sessions) and paresthesia (1.1%), which were probably due to citrate anticoagulation. Access problems had to be overcome in 1.5%, adsorber and hardware problems in 0.5% of the sessions. In this multicenter long‐term study, DALI apheresis proved to be an efficient, safe, and easy procedure for extracorporeal LDL and Lp(a) elimination. J. Clin. Apheresis 17:161–169, 2002. © 2002 Wiley‐Liss, Inc.
Fuji surge technique and continuous in‐line filtration to improve the quality of single donor platelet concentrates
Authors: Moog R.
Published: December 2002

White blood cell (WBC)‐reduced single donor platelet concentrates (SDPs) can be collected by most cell separators. WBC reduction can be achieved directly during plateletpheresis or by filtration. Continuous filtration with low filtration rates provides SDPs of good purity. To compensate the platelet (PLT) loss due to filtration, the PLT yield in the unfiltered primary product should be optimal. Fifty donors underwent plateletpheresis with the MCS+ blood cell separator (Haemonetics) with the new Fuji surge technique and continuous WBC filtration. Twelve SDPs were analysed for PLT yield, red blood cells (RBC), WBC, and pH after collection (Day 0) and at the end of storage (Day 5). Thereafter, further 38 SDPs were measured for PLT and WBC content in routine production at Day 0. PLT were determined electronically, RBC and WBC were counted manually (Neubauer and Nageotte chamber, respectively). For pH measurement, a pH‐meter was used. Mean blood volume processed was 2,621 ± 112 ml in a donation time of 76 ± 10 min. An average PLT yield of 3.45 ± 0.88 × 1011 was collected in a product volume of 325 ± 77 ml. The collection efficiency was 60.0 ± 5.5%. WBC contamination of all units tested was 0.046 ± 0.059 × 106 and the RBC content of the SDPs analysed at Day 0 was 0.014 ± 0.003 × 109. The pH was well maintained over the storage period of 5 days. The data indicate that Fuji surge technique and continuous in‐line leukocyte filtration allow for the collection of SDPs with high platelet yield and low leukocyte contamination, meeting the Council of Europe quality standards. J. Clin. Apheresis 17:199–203. © 2002 Wiley‐Liss, Inc.
Molecular cloning of Drosophila HCF reveals proteolytic processing and self‐association of the encoded protein
Authors: Mahajan Shahana S. , Johnson Kristina M. , Wilson Angus C.
Published: December 2002

HCF‐1 functions as a coactivator for herpes simplex virus VP16 and a number of mammalian transcription factors. Mature HCF‐1 is composed of two subunits generated by proteolytic cleavage of a larger precursor at six centrally‐located HCFPRO repeats. The resulting N‐ and C‐terminal subunits remain tightly associated via two complementary pairs of self‐association domains: termed SAS1N‐SAS1C and SAS2N‐SAS2C. Additional HCF proteins have been identified in mammals (HCF‐2) and Caenorhabditis elegans (CeHCF). Both contain well‐conserved SAS1 domains but do not undergo proteolytic processing. Thus, the significance of the cleavage and self‐association of HCF‐1 remains enigmatic. Here, we describe the isolation of the Drosophila HCF homologue (dHCF) using a genetic screen based on conservation of the SAS1 interaction. The N‐terminal β‐propeller domain of dHCF supports VP16‐induced complex formation and is more similar to mammalian HCF‐1 than other homologues. We show that full‐length dHCF expressed in Drosophila cells undergoes proteolytic cleavage giving rise to tightly associated N‐ and C‐terminal subunits. As with HCF‐1, the SAS1N and SAS1C elements of dHCF are separated by a large central region, however, this sequence lacks obvious homology to the HCFPRO repeats required for HCF‐1 cleavage. The conservation of HCF processing in insect cells argues that formation of separate N‐ and C‐terminal subunits is important for HCF function. © 2002 Wiley‐Liss, Inc.
Gαq signaling is required for Rho‐dependent transcriptional activation of the cyclooxygenase‐2 promoter in fibroblasts
Authors: Slice Lee W. , Han Sang‐Kyou , Simon Melvin I.
Published: December 2002

Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase‐2 (COX‐2) expression through a Rho‐dependent, protein kinase C (PKC)‐independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562–27566). However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G‐proteins) that are coupled to the GRP receptor in Rho‐dependent COX‐2 expression has not been elucidated. In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock‐outs (DKO) for Gαq/11 and Gα12/13 to demonstrate that COX‐2 promoter activation by GRP requires Gαq. Furthermore, we show that GRP‐dependent COX‐2 gene expression, as assessed by a COX‐2 reporter luciferase assay, was induced in cells lacking Gα12/13 but was blocked in cells that did not express Gαq/11. GRP‐dependent COX‐2 promoter induction in Gαq/11 deficient cells was rescued by expression of wild type Gαq but blocked by inhibition of calcium signaling in calcium‐free media or in cells treated with 2‐aminoethoxydiphenylborate (2‐APB). Co‐stimulation of transfected Gαq/11 deficient cells with GRP and thapsigargin (TG) induced the COX‐2 promoter. Activation of endogenous Rho by expression of Onco‐lbc or expression of Rho A Q63L resulted in COX‐2 promoter activation in Gαq/11 deficient cells. Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX‐2 promoter induction. Expression of Gαq Q209L in the well‐characterized fibroblast cell line, NIH3T3, induced the COX‐2 promoter which was blocked by expression of C3 toxin. These results demonstrate that calcium signaling mediated by Gαq and Rho play critical roles in GRP‐dependent COX‐2 expression in fibroblasts. © 2002 Wiley‐Liss, Inc.
Retinoic acid‐induced AP‐1 transcriptional activity regulates B16 mouse melanoma growth inhibition and differentiation
Authors: Huang Ying , Boskovic Goran , Niles Richard M.
Published: December 2002

Retinoic acid (RA) inhibits growth and induces differentiation of B16 mouse melanoma cells. These effects are accompanied by a large increase in PKCα mRNA and protein levels and surprisingly an increase in activating protein‐1 (AP‐1) transcriptional activity. To further investigate the RA‐induced AP‐1 activity we established clones of B16 cells stably expressing an AP‐1‐luciferase reporter gene. Treatment of these clones with phorbol dibutyrate increased AP‐1 activity which peaked at 2–4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP‐1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. We tested the importance of the RA‐induced AP‐1 activity by establishing clones which stably express a dominant negative fos gene (A‐fos) and have greatly diminished AP‐1 activity. Growth rates of untreated A‐fos expressing cells were similar to wt B16 and clones not expressing A‐fos. However, clones expressing the dominant‐negative fos had a markedly decreased sensitivity to RA‐induced inhibition of anchorage‐dependent and ‐independent growth. Treatment of wt B16 cells for 48 h with RA increased melanin production by two to fourfold, but this effect was completely lost in the A‐fos clones. The ability of RA to induce RARβ and PKCα expression was retained in A‐fos clones, suggesting that A‐fos was not interfering with RAR transcription activation functions. We tested whether the RA‐induced AP‐1 activity might be mediated by the ERK1/2 MAPK pathway. Inhibition of ERK1/2 phosphorylation stimulated AP‐1 activity, which was not additive to that induced by RA. This finding raises the possibility that this MAPK pathway may be a target of retinoid action. Our observations suggest that AP‐1 transcriptional activity induced by RA likely plays an important role in the biological changes mediated by this retinoid in B16 melanoma cells. © 2002 Wiley‐Liss, Inc.
Differential levels of soluble endoglin (CD105) in myeloid malignancies
Authors: Calabrò Luana , Fonsatti Ester , Bellomo Giacomo , Alonci Andrea , Colizzi Francesca , Sigalotti Luca , Altomonte Maresa , Musolino Caterina , Maio Michele
Published: December 2002

Angiogenesis contributes to disease progression in solid and hematopoietic malignancies, and endoglin (CD105), a component of the transforming growth factor (TGF)‐β receptor complex, is a powerful marker of neovascularization. Elevated amounts of soluble CD105 (sCD105) have been recently identified in selected solid tumors but no data are available on sCD105 in hematopoietic malignancies. Therefore, levels of sCD105 were investigated in sera of patients with acute myeloid leukemia (AML) (n = 10) or chronic myeloproliferative disorders (CMD) (n = 28), and correlated with those of soluble TGF‐β1 (sTGF‐β1). Dot blot assay detected higher amounts of sCD105 (P < 0.05) both in AML (4.34 ± 2.62 OD/mm2) and in CMD (3.71 ± 2.09 OD/mm2) patients than in healthy subjects (n = 14, 2.38 ± 1.18 OD/mm2). Instead, enzyme‐linked immunosorbent assay (ELISA) identified (P < 0.05) lower and higher levels of sTGF‐β1 in AML (32,017 ± 1,900 pg/ml) and CMD (60,700 ± 19,200 pg/ml) patients, respectively, compared to healthy individuals (n = 11, 47,173 ± 5,443 pg/ml). In essential thrombocythemia (ET) patients with thrombotic episodes, levels of sCD105 were lower (P < 0.05) compared to patients without thrombotic complications, and inversely correlated with those of sTGF‐β1 (r = 0.94). Conversely, amounts of sCD105 directly correlated with levels of sTGF‐β1 (r = 0.74) in ET patients without thrombotic events. Our results show that high levels of sCD105 are present in myeloid malignancies that are characterized by a high cellular proliferation rate, and suggest that an altered balance between sCD105 and sTGF‐β1 might favor disease progression and clinical complications. © 2002 Wiley‐Liss, Inc.
Role of the IGF‐I receptor in the regulation of cell–cell adhesion: Implications in cancer development and progression
Authors: Mauro Loredana , Salerno Michele , Morelli Catia , Boterberg Tom , Bracke Marc E. , Surmacz Eva
Published: December 2002

The insulin‐like growth factor‐I receptor (IGF‐IR) is a ubiquitous multifunctional tyrosine kinase that has an important role in normal cell growth and development. However, abnormal stimulation of IGF‐IR signaling has been implicated in the development of different types of tumors. The strong antiapoptotic activity of IGF‐IR has been recognized as critical in IGF‐I‐dependent tumorigenesis, however, the impact of other IGF‐IR functions, such as regulation of cell–cell and cell–matrix adhesion are also increasingly acknowledged. Here, on the model of breast cancer cells, we discuss how IGF‐IR‐dependent regulation of intercellular adhesion may affect cell survival, resistance to antiestrogens, and invasion. © 2002 Wiley‐Liss, Inc.
HOX gene network is involved in the transcriptional regulation of in vivo human adipogenesis
Authors: Cantile Monica , Procino Alfredo , D'armiento Maria , Cindolo Luca , Cillo Clemente
Published: December 2002

Adipogenesis is regulated by the sequential activation of a series of transcription factors: the C/EBP proteins of type β and δ trigger the process while PPARγ and C/EBPα induce the differentiation from pre‐adipocyte to adipocyte, followed by adipo‐specific gene expression. A number of observations suggest the involvement of genes controlling embryonal development in adipogenesis. In human thyroid follicular carcinoma, it has been recently identified an oncogenetic fusion protein resulting from the interaction between the isoform PPARγ1 of PPARγ and the homeoprotein encoded by the PAX‐8 gene. Recent observations have pointed out that gene expression associated with adipocyte differentiation in vivo and in vitro, although partially overlapping, is actually different. HOX genes make up a network of transcription factors (homeoproteins) controlling embryonal development as well as crucial functions of adult eukaryotic cells. The molecular organization of this network of 39 genes appears to be unique in the genome and probably acts regulating phenotypic cell identity. In the present study we have analyzed the expression of the complete HOX gene network, in vivo, in different deposits of human white adipose tissue and in embryonal brown adipose tissues. Most of the genes in the HOX network are active in white as well as brown adipose tissue. Furthermore HOX genes display a deposit‐specific expression in white adipose tissue. Moreover, expression of the paralogous group 4 genes (HOX A4, HOX B4, HOX C4, and HOX D4), together with that of isolated genes in the network, appears to discriminate between white and brown adipose tissue. This data allows us to postulate the involvement of the HOX network in transcriptional regulation of human adipogenesis and to hypothesize on the molecular mechanisms that could be implicated. © 2002 Wiley‐Liss, Inc.
Fluid shear stress inhibits TNF‐α‐induced apoptosis in osteoblasts: A role for fluid shear stress‐induced activation of PI3‐kinase and inhibition of caspase‐3
Authors: Pavalko Fredrick M. , Gerard Rita L. , Ponik Suzanne M. , Gallagher Patricia J. , Jin Yijun , Norvell Suzanne M.
Published: December 2002

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3‐E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF‐α in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4–6 h of exposure to TNF‐α and CHX, and application of FSS (12 dyne/cm2) significantly attenuated this TNF‐α‐induced apoptosis. FSS activated PI3‐kinase signaling, induced phosphorylation of Akt, and inhibited TNF‐α‐induced activation of caspase‐3. Inhibition of PI3‐kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF‐α‐induced apoptosis and blocked FSS‐induced inhibition of caspase‐3 activation in osteoblasts treated with TNF‐α. LY294002 did not, however, prevent FSS‐induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3‐kinase‐independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF‐α‐induced apoptosis through a mechanism involving activation of PI3‐kinase signaling and inhibition of caspases. FSS‐induced activation of PI3‐kinase may promote cell survival through a mechanism that is distinct from the Akt‐mediated survival pathway. © 2002 Wiley‐Liss, Inc.
SNARE protein degradation upon platelet activation: Calpain cleaves SNAP‐23
Authors: Lai Katharine Crane , Flaumenhaft Robert
Published: December 2002

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation‐induced degranulation on three functional platelet SNARE proteins, SNAP‐23, VAMP‐3, and syntaxin 4. Initial studies showed that SNAP‐23 is lost upon SFLLRN‐induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP‐23 was Ca2+‐dependent. Ca2+‐dependent proteolysis of SNAP‐23 was inhibited by the cell‐permeable calpain inhibitors, calpeptin and E‐64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP‐23 in permeabilized platelets in a dose‐dependent manner. In intact platelets, calpeptin prevented SFLLRN‐induced degradation of SNAP‐23. In contrast, calpeptin did not prevent SFLLRN‐induced degradation of VAMP‐3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain‐induced cleavage of SNAP‐23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca2+. Experiments evaluating platelet α‐granule secretion demonstrated that incubation of permeabilized platelets with 10 μM Ca2+ prior to exposure to ATP inhibited ATP‐dependent α‐granule secretion from permeabilized platelets. SNAP‐23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca2+‐mediated degradation of SNAP‐23 and reversed Ca2+‐mediated inhibition of ATP‐dependent α‐granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP‐23 and inhibits α‐granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation. © 2002 Wiley‐Liss, Inc.
Quantitative and qualitative adaptation of skeletal muscle mitochondria to increased physical activity
Authors: Zoll Joffrey , Koulmann Nathalie , Bahi Lahoucine , Ventura‐Clapier Renée , Bigard André‐Xavier
Published: December 2002

Endurance capacity rely on high muscle oxidative capacity but should also involve a tighter coupling between energy production and utilization within the myocyte. The present study examined the responses of muscle oxidative capacity and the regulation of oxidative phosphorylation by phosphate acceptors in locomotor muscles of voluntary running rats (n = 8), using saponin permeabilized fibers of the deep and superficial parts of plantaris muscle (dPLA and sPLA, respectively). Non‐ADP stimulated respiration of skinned fibers increased by 33% (P < 0.05) and 100% (P < 0.001) in sPLA and dPLA, respectively. The maximal ADP‐stimulated respiration was 57% (P < 0.001) and 32% (P < 0.01) higher in active rats than in sedentary rats (n = 8), in sPLA and dPLA, respectively. This finding was consistent with a 72% increase in the CS activity in plantaris muscle of exercising rats (P < 0.01). Voluntary running induced a 334% increase in the apparent Km for ADP in sPLA (P < 0.001), and a 61% increase in dPLA (P < 0.05), showing a lower affinity for cytosolic ADP of mitochondria present in both, predominantly glycolytic, and oxidative fibers. There was an increase in the creatine kinase efficacy in both sPLA and dPLA (131%, 75%, P < 0.001, respectively), consistent with an increase in the activity of the mitochondrial isoform of creatine kinase (106%, P < 0.01). It is concluded that, in addition to the well‐known increased oxidative capacity, voluntary running is associated with changes in the regulation of oxidative phosphorylation by phosphate acceptors, in both glycolytic and oxidative fibers, in the direction of increased coupling between energy production and energy utilization. © 2002 Wiley‐Liss, Inc.
Current Literature in Journal of Mass Spectrometry
Authors:   John Wiley & Sons, Ltd.
Published: December 2002

In order to keep subscribers up‐to‐date with the latest developments in their field, John Wiley & Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of mass spectrometry. Each bibliography is divided into 11 sections: 1 Books, Reviews & Symposia; 2 Instrumental Techniques & Methods; 3 Gas Phase Ion Chemistry; 4 Biology/Biochemistry: Amino Acids, Peptides & Proteins; Carbohydrates; Lipids; Nucleic Acids; 5 Pharmacology/Toxicology; 6 Natural Products; 7 Analysis of Organic Compounds; 8 Analysis of Inorganics/Organometallics; 9 Surface Analysis; 10 Environmental Analysis; 11 Elemental Analysis. Within each section, articles are listed in alphabetical order with respect to author (4 Weeks journals ‐ Search completed at 18th. Sept. 2002)
A new external EI/CI source–ion trap system devoted to the study of ion‐molecule reactions
Authors: Pace Giuseppe , Seraglia Roberta , Cacace Fulvio , Petris Giulia de , Traldi Pietro
Published: December 2002

A new instrumental arrangement dedicated to the study of ion‐molecule reactions and consists of by an electron impact/chemical ionization source mounted 420 mm away from an ion trap is described. It has been designed and developed to exclude diffusion into the ion trap of the neutral reactants from the EI/CI source and so to avoid undesired side‐reactions. Its instrumental parameterization is described and some preliminary results are illustrated that show promise for the successful application of the system to the study of ion‐molecule reactions. Copyright © 2002 John Wiley & Sons, Ltd.
Gas‐phase ion chemistry in the ternary SiH4–C3H6–NH3 system
Authors: Calderan Stefania , Carbone Paola , Operti Lorenza , Rabezzana Roberto , Vaglio Gian Angelo
Published: December 2002

The gas‐phase ion chemistry of propene–ammonia and silane–propene–ammonia mixtures was studied by ion trap mass spectrometry. As far as the binary mixture is concerned, the effect of different molar ratios of the reactants on the trend of ion species formed was evaluated, the ion–molecule reaction processes were identified and the rate constants for the main processes were measured. The results were compared with the collisional rate constants to determine the reaction efficiencies. In the ternary silane–propene–ammonia mixture the mechanisms of formation of SimCnNpHq+ clusters were elucidated and the rate constants of the most important steps were measured. For some species, selected by double isolation (MS/MS), the low abundance of the ions allowed us to determine the reaction paths but not the rate constants. Ternary ions are mainly formed by reactions of SimCnHq+ ions with ammonia, whereas a minor contribution comes from reactions of SimNpHq+ ions with propene. On the other hand, the CnNpHq+ ions showed a very low reactivity and no step leading to ternary ion species was identified. The formation of hydrogenated ternary ions with Si, C and N has a basic importance in relation to their possible role as precursors of amorphous silicon carbides doped with nitrogen obtained by deposition from silane–propene–ammonia mixtures properly activated. Copyright © 2002 John Wiley & Sons, Ltd.
Capillary electrophoresis/mass spectrometry: a promising tool for the control of some physiologically hazardous compounds. I—Derivatives of 3‐quinuclidinol
Authors: Bednář Petr , Lemr Karel , Barták Petr , Ševčík Juraj , Hlaváč Jan , Stýskala Jakub , Wiedermannová Iveta , Stránský Zdeněk
Published: December 2002

A method based on the coupling of capillary electrophoresis with mass spectrometry (CE/MS) was developed for the monitoring of 3‐quinuclidinol and its four N‐alkyl derivatives (methyl, ethyl, propyl and isopropyl derivatives). A fragmentation study (collision‐induced dissociation of ions in an ion trap) and optimization of the ion optics set‐up for CE/MS experiments using direct infusion of a methanolic solution of the standards into the mass spectrometer were carried out in advance. Molecular ions of all quaternary compounds and the quasi‐molecular ion [M + H]+ of free 3‐quinuclidinol prevail in the mass spectra. In the MS/MS of propyl and isopropyl derivatives, the elimination of the alkyl chain dominates, leading to the ion at m/z 128. The fragmentation of the other compounds is more complex. Previous CE separation of the mixture of isobaric propyl and isopropyl derivatives is necessary for their unambiguous identification. A 10 mM ammonium acetate buffer (pH 4.0) is the optimum running electrolyte, allowing the CE separation of methyl, ethyl, propyl and isopropyl derivatives. A 0.5% (v/v) solution of acetic acid in methanol provides sufficient detection sensitivity when used as the sheath liquid. Limits of detection of 0.1 ppm for 3‐quinuclidinol and 0.05 ppm for quaternary derivatives were achieved under the optimum conditions. The optimized method was applied to the determination of 3‐quinuclidinol and related quaternary derivatives spiked into a sample of pond water. The experimental set‐up for CE/MS/MS was investigated, which strongly increases the identification capability of the technique. Copyright © 2002 John Wiley & Sons, Ltd.
Detoxification of cytotoxic α,β‐unsaturated aldehydes by carnosine: characterization of conjugated adducts by electrospray ionization tandem mass spectrometry and detection by liquid chromatography/mass spectrometry in rat skeletal muscle
Authors: Aldini Giancarlo , Granata Paola , Carini Marina
Published: December 2002

Oxidation of polyunsaturated fatty acids containing phospholipids in tissue generates lipid hydroperoxides, which are further degraded to several products, among which unsaturated aldehydes such as 4‐hydroxy‐trans‐2‐nonenal (HNE) play an important role in mediating the pathological effects of oxidative stress. While the reaction of HNE with glutathione (GSH) is a well recognized pathway of detoxification in biological systems, no data are available on HNE interactions with carnosine, a dipeptide (β‐alanyl‐L‐histidine) present in high concentration in skeletal muscle. The aim of this work was to study the quenching ability of carnosine towards HNE and to characterize the reaction products by electrospray ionization tandem mass spectrometry (ESI‐MS/MS), using GSH as a model peptide. GSH incubation with HNE in 1 mM phosphate buffer (pH 7.4) results in the complete disappearance of HNE within 1 h owing to the formation of a Michael adduct, S‐(4‐hydroxynonanal‐3‐yl)glutathione. The reaction of HNE with carnosine was studied in different molar ratios and monitored up to 24 h by high‐performance liquid chromatography (HPLC) (HNE consumption), MS/MS (infusion) and liquid chromatography mass spectrometry (LC/MS) experiments. Carnosine, although less reactive than GSH, significantly quenched HNE (48.2 ± 0.9% HNE consumption after 1 h; carnosine:HNE molar ratio 10 : 1). Two reaction products were identified: the Michael adduct, N‐(4‐hydroxynonanal‐3‐yl)carnosine involving the imidazolic nitrogen of histidine, and the imine adduct, involving the amino group of the β‐alanine residue. Definitive structure assignment was achieved by chemical reduction with NaBH4 and multinuclear magnetic resonance experiments. To understand whether carnosine acts as a quencher of unsaturated aldehydes in biological matrices, rat skeletal muscle homogenate was incubated with HNE and the formation of conjugated adducts was determined by LC/MS analysis. Three main products were detected and identified as Michael adducts of HNE with GSH, carnosine and anserine (the N‐methylated derivative of carnosine, present in high concentrations in rat muscle). The results indicate that beside GSH, histidine‐containing dipeptides could be involved in the detoxification pathway of reactive aldehydes from lipid peroxidation generated in skeletal muscle during physical endurance. Copyright © 2002 John Wiley & Sons, Ltd.
Solid‐phase microextraction gas chromatography/ion trap mass spectrometry and multistage mass spectrometry experiments in the characterization of germacrene D
Authors: Gioacchini A. M. , Menotta M. , Polidori E. , Giomaro G. , Stocchi V.
Published: December 2002

Germacrene D is a vegetable pheromone utilized in interactions among organisms belonging to different species. For the first time, using solid‐phase microextraction/gas chromatography/ion trap mass spectrometry, the presence of this compound was detected in an in vitro mycorrhizal synthesis system where the mycelium of the ectomycorrhizal fungus Tuber borchii Vittad. interacts with the plant Tilia Americana L. From this symbiosis, a new structure, called ectomycorrhiza, is formed where the two symbionts exchange nutrients and metabolites. It seems that only after this interaction can the mycelium develop the fruitbody, commonly known as truffle. The results obtained allowed us to ascertain that germacrene D was synthesized by the plant exclusively in the presence of T. borchii. The originality of these data prompted us to hypothesize that this compound could be involved in the first step of ectomycorrhiza formation, as it is able to stimulate specific fungi receptors. In fact, plants release hundreds of secondary metabolites that are important in their interactions with other organisms. Copyright © 2002 John Wiley & Sons, Ltd.