bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐resolution 19p13.2–13.3 allelotyping of breast carcinomas demonstrates frequent loss of heterozygosity
Authors: Yang Tseng‐Long , Su Yen‐Rey , Huang Chiun‐Sheng , Yu Jyh‐Cherng , Lo Yen‐Li , Wu Pei‐Ei , Shen Chen‐Yang
Published: August 2004

In breast cancer, a high frequency of genomic deletion is found in chromosomal region 19p13. Of particular interest is that the LKB1 gene (also known as STK11) has been mapped to this region. LKB1 is responsible for Peutz–Jeghers syndrome (PJS), a genetic disease characterized by mucocutaneous pigmentation and gastrointestinal hematoma with an increased risk of developing cancer, including breast cancer. To further clarify the role of chromosomal region 19p13.2–13.3 in the pathogenesis of breast cancer and to identify more precisely candidate tumor‐suppressor genes (TSGs) for positional cloning studies, we performed detailed high‐resolution allelotyping analysis to detect allelic loss or loss of heterozygosity (LOH) in this region on microdissected samples from 140 primary breast tumors using 24 microsatellite markers. The highest frequencies of LOH were seen with D19S883 (30%) and D19S216 (29%), both at 19p13.3, D19S922 (28%), at 19p13.3–19p13.2, and D19S865 (39%), at 19p13.2; in addition, identification was made of at least four common deletion regions, including the LKB1 locus, that are centered on these four markers. In all the cases, we found discontinuous allele loss at several 19p13.2–13.3 sites in the same tumor (with the markers with the highest frequency of LOH adjacent to markers retaining heterozygosity), suggesting the presence of multiple TSGs. Interestingly, in tumors, the extent of allelic loss at these markers (measured as the fractional allele loss) increased significantly as the tumors progressed to poorer grades (P < 0.05). We conclude that 19p13.2–13.3 allele loss is a common event in the pathogenesis of breast carcinoma that often involves discontinuous LOH of multiple, localized TSGs (including LKB1), the concurrent inactivation of which may contribute to breast cancer progression. © 2004 Wiley‐Liss, Inc.
Chromosome band 16q22‐linked familial AML: Exclusion of candidate genes, and possible disease risk modification by NQO1 polymorphisms
Authors: Escher Robert , Jones Antonia , Hagos Fitsum , Carmichael Catherine , Horwitz Marshall , Olopade Olufunmilayo I. , Scott Hamish S.
Published: August 2004

Analyses of chromosomal translocation and inversion breakpoints in sporadic acute myeloid leukemias have identified many transcription factors as playing a role in leukemogenesis. Studies of families with a Mendelian predisposition to hematological malignancies have identified the gene coding for the transcription factor RUNX1 as a leukemia‐predisposing gene involved in the first steps of leukemogenesis. Using two families, another autosomal dominant familial leukemia locus was linked to chromosome band 16q22 where the CBFB gene maps. Although CBFB forms a core‐binding factor transcriptional complex with RUNX1, previous analyses have excluded the CBFB gene as the leukemia‐predisposing gene in these families. In the current study, we performed an extended molecular analysis in these families of the four other transcription factor genes in the 16q22 critical region as well as of two other genes with a known association with leukemia. Several previously undescribed but nonpathogenic sequence variants were identified. We demonstrated that the transcription factors E2F4, CTCF, NFATC3, and NFAT5, and the genes coding for NAD(P)H:quinone oxido‐reductase 1 (NQO1) and for E‐cadherin are not responsible for the leukemia susceptibility in these families. The presence of NQO1 polymorphisms may suggest a role for this gene in disease risk modification in these families. © 2004 Wiley‐Liss, Inc.
Comparative expressed sequence hybridization studies of high‐hyperdiploid childhood acute lymphoblastic leukemia
Authors: Gruszka‐Westwood Alicja M. , Horsley Sharon W. , Martinez‐Ramirez Angel , Harrison Christine J. , Kempski Helena , Moorman Anthony V. , Ross Fiona M. , Griffiths Michael , Greaves Mel F. , Kearney Lyndal
Published: August 2004

The functional consequences of a high‐hyperdiploid karyotype, found in up to one‐third of cases of acute lymphoblastic leukemia (ALL), are unknown. Using the technique of comparative expressed sequence hybridization (CESH), we sought to address the question of whether increased chromosome copies in hyperdiploid ALL lead to increased gene expression. Relative expression of hyperdiploid ALL blasts versus peripheral blood mononuclear cells was analyzed in 18 patients. Common regions of overexpression corresponding to the presence of tri‐/tetrasomies included: Xp22.1–22.2, 4q28, 6q14–15, 6q24, 10p13, 14q23–24, 17q21, 18q12, and 21q21, identified in 28–89% of cases. However, increased expression without underlying trisomy occurred at 3p21.3, 7q11.2, 8p21, and 8q24.1 in 39–90% of cases. High expression at 7q11.2, the most consistent change detected, was confirmed by quantitative PCR. Poor correlation between the presence of tri‐/tetrasomy and overexpression was observed for chromosomes 14 and 17. Two cases were reanalyzed versus (i) B cells, (ii) transformed B cells, and (iii) CD34+19+ cells (the putative counterpart of the leukemic cell). A reduction in the number of relatively overexpressed regions was observed with CD34+19+ cells. In particular, the peak at 7q11.2 disappeared, suggesting up‐regulation of genes from this region in the early ontology of normal B‐cell development. In conclusion, we have shown that tri‐/tetrasomies in hyperdiploid ALL lead to an increase in the expression of associated sequences. The choice of a biologically relevant reference is crucial for data interpretation. © 2004 Wiley‐Liss, Inc.
Molecular cytogenetic characterization of proximal‐type epithelioid sarcoma
Authors: Lualdi Elena , Modena Piergiorgio , Debiec‐Rychter Maria , Pedeutour Florence , Teixeira Manuel R. , Facchinetti Federica , Dagrada Gian Paolo , Pilotti Silvana , Sozzi Gabriella
Published: August 2004

Proximal‐type epithelioid sarcoma is a recently described soft‐tissue tumor that is distinguished from conventional‐type epithelioid sarcoma by a far more aggressive clinical course, frequent location in the proximal anatomic regions, and variable rhabdoid morphology. Because of their rarity and peculiar morphology, proximal‐type epithelioid sarcomas frequently pose serious diagnostic dilemmas, being easily misdiagnosed as a variety of other malignant neoplasms. To date, the information available on the genetic alterations associated with this tumor entity has been confined to single conventional cytogenetic reports. In this article, we present the results of a conventional and molecular cytogenetic analysis of six proximal‐type epithelioid sarcomas. Spectral karyotyping analysis of these cases deciphered the characteristics of several marker chromosomes and complex translocations, leading to the recognition of recurrent rearrangements. The most frequently involved chromosome arm was 22q, and the identification of two cases with a similar translocation, t(10;22), suggests a role for one or more genes on chromosome 22 in the pathogenesis of this tumor and provides an opportunity for finely mapping the translocation‐associated breakpoints. Chromosome arm 8q gain was also a frequent event and correlated with gain of MYC gene copy number, as demonstrated by fluorescence in situ hybridization. A review of both cases reported in the literature and those presented in this study reinforced the involvement of chromosomes 8 and 22 and also indicated frequent rearrangements of chromosomes 7, 14, 18, and 20. © 2004 Wiley‐Liss, Inc.
Structural integrity and expression of the L3MBTL gene in normal and malignant hematopoietic cells
Authors: MacGrogan Donal , Kalakonda Nagesh , Alvarez Sara , Scandura Joseph M. , Boccuni Piernicola , Johansson Bertil , Nimer Stephen D.
Published: August 2004

The human L3MBTL gene is located in 20q12, a region that is commonly deleted in myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). L3MBTL is highly homologous to the D‐lethal(3) malignant brain tumor [D‐l(3)mbt] gene, which is a putative tumor‐suppressor gene (TSG) identified in Drosophila and which is closely related to the Drosophila sex combs on midleg (SCM) protein, a member of the Polycomb group (PcG) family of transcriptional repressors. To examine whether L3MBTL functions as a “classic” TSG in human hematologic malignancies, we screened a panel of 17 myeloid leukemia cell lines and peripheral blood or bone marrow samples from 29 MDS and 13 MPD patients for mutations in the entire L3MBTL coding sequence, including intron/exon splice junctions. No mutations were identified, although two single nucleotide differences were found (in intron 14 and in exon 15), which were interpreted as polymorphic changes. We used real‐time RT‐PCR to quantify the level of L3MBTL mRNA in various normal myeloid and lymphoid cell populations. L3MBTL is expressed in normal CD34+ bone marrow cells, and we found that the pattern of L3MBTL expression was similar to that of BMI1, a well‐studied PcG gene with oncogenic activity, suggesting that L3MBTL and BMI1 may be co‐regulated during hematopoiesis. The expression of L3MBTL mRNA in 30 of 35 cell lines and 13 of 15 AML samples was comparable to the level of L3MBTL expression in the normal cell populations. However, five leukemia cell lines showed no L3MBTL expression, and two of the AML samples showed aberrant L3MBTL expression. These data suggest that L3MBTL is not mutated in MDS or MPD. However, given the known dosage effects of PcG proteins in regulating gene expression, reduced or absent L3MBTL expression may be relevant in some cases of myeloid leukemia. © 2004 Wiley‐Liss, Inc.
Various types of rearrangements target TLX3 locus in T‐cell acute lymphoblastic leukemia
Authors: Su Xin Ying , Busson Maryvonne , Della Valle Véronique , Ballerini Paola , Dastugue Nicole , Talmant Pascaline , Ferrando Adolfo A. , Baudry‐Bluteau Dominique , Romana Serge , Berger Roland , Bernard Olivier A.
Published: August 2004

Most chromosomal translocations observed in T‐cell acute lymphoblastic leukemia (T‐ALL) often produce transcriptional activation of transcription factor oncogenes. Ectopic expression of the TLX3 (also known as HOX11L2) gene has been shown to be associated with a cryptic t(5;14)(q35;q32) translocation specific for a subtype of T‐ALL. Here we report several examples of variant and alternative translocations resulting in expression of TLX3 in T‐ALL, and we describe three of these translocations in detail. In particular, the CDK6 gene was rearranged in two t(5;7)(q35;q21) translocations. In two additional instances, fusion of the BCL11B (also known as CTIP2) and RANBP17/TLX3 loci were shown to result from subtle genomic insertion/deletion within these loci. This study further underscores that TLX3 expression in T‐ALL is strongly associated with the presence of genomic rearrangements. © 2004 Wiley‐Liss, Inc.
Aberrations of breast cancer susceptibility genes occur early in sporadic breast tumors and in acquisition of breast epithelial immortalization
Authors: Meng Zhen Hang , Ben Yong , Li Zheng , Chew Karen , Ljung Britt‐Marie , Lagios Michael D. , Dairkee Shanaz H.
Published: August 2004

In the search for early deletion targets in sporadic breast cancer, the analysis of TP53, BRCA1, BRCA2, and ATM revealed loss of heterozygosity (LOH) in tumor cells at 1 or more genes in 18 of 24 cases examined. Notably, in more than 60% of such tumors, LOH was detectable in morphologically normal terminal ductal lobular units (TDLUs) adjacent to carcinoma (LOHint). At BRCA2 and ATM, LOHint was most frequent, particularly in TDLUs of women ≤ 50 years old (7 of 10). In a novel preneoplastic model system, declining expression levels observed with increasing passage in culture supported the postulate that during the acquisition of continuous growth, elimination of these genes at an early stage confers a distinct selective advantage. The intimate role of these genes in DNA repair and their early deletion has implications for the possible transforming effects of DNA‐damaging agents on unexcised breast tissue harboring LOHint within breast cancer patients. © 2004 Wiley‐Liss, Inc.
Astrocytic exocytosis vesicles and glutamate: A high‐resolution immunofluorescence study
Authors: Anlauf Enrico , Derouiche Amin
Published: August 2004

Physiological evidence has demonstrated that cultured astrocytes can release glutamate via Ca2+‐dependent mechanisms. Also, glutamate released from astrocytes in the hippocampal slice interferes with synaptic neurotransmission. Since these observations suggest vesicular glutamate release from astrocytes, the presence of glutamate‐containing exocytosis vesicles was investigated. We applied immunofluorescence techniques combined with high‐performance deconvolution microscopy, which yields a resolution of <200 nm and permits evaluation of double labeling in individual vesicles. Using a well‐characterized anti‐glutamate antiserum and parameters minimizing fixative‐induced autofluorescence, glutamate‐immunoreactive (ir) puncta were found all over the astrocyte but were conspicuously dense at the cell boundary and in filopodia. Images were very similar with antibodies against vesicular glutamate transporters (vGluT1 and vGluT2). Labeling for the exocytosis markers rab3, synaptophysin, or synaptobrevin was also punctate, particularly dense at the cell boundary, but disappearing toward the perinuclear region. Sections of the cell boundary were delineated by rab3 immunoreactivity. In double‐labeled cells, vesicular colocalization of glutamate and any of the exocytosis markers was frequent in filopodia and at the cell boundary. Within the cell, single‐labeled glutamate‐ir vesicles prevailed; double‐labeled vesicles were infrequently present. By resolving single vesicles, in cultured astrocytes we visualize glutamate‐containing vesicles, vesicles displaying vGluT1 or vGluT2, and exocytosis vesicles displaying glutamate‐ir. This may provide the morphological correlate of Ca2+‐dependent glutamate release from astrocytes, possibly occurring at defined sections of the cell membrane and at filopodia. However, since vGluTs and exocytosis markers are classically restricted to nerve terminals in the CNS, glutamate release from astrocytes in the CNS remains to be studied. © 2004 Wiley‐Liss, Inc.
Root growth and soil water utilization of winter wheat in the North China Plain
Authors: Zhang Xiying , Pei Dong , Chen Suying
Published: August 2004

Winter wheat (Triticum aestivum L.) is the major crop in the North China Plain (NCP). The monsoon climate in this region causes most rain to fall in the summer season, but during the winter‐wheat growing season (October–May) the rainfall is far less than the water requirements for the crop. The efficiency of soil water use by winter wheat needs to be improved to reduce the need for irrigation. In this paper, we report the results of two seasons' work on soil water utilization, root growth and distribution, root water uptake by the crop under different irrigation treatments, and possible ways to improve soil water‐use efficiency. The field experiments were carried out at Luancheng Station (37° 53′N, 114° 41′E) from 1996 to 1997 and 1998 to 1999, two growing seasons of winter wheat. Five treatments for each season: rain‐fed and irrigated winter wheat with different irrigation numbers from 1 up to 4, were set up in a randomized plot design. Soil available water‐holding capacity at the experimental site was about 454 mm for the top 2 m soil profile. Root sampling results showed that winter wheat had a prolific root system with an average maximum rooting depth of 2 m. Most of the root system was concentrated in the upper 40 cm of soil. Root length density in the top layer of soil (0–20 cm) was very high, with values over 5 cm cm−3. The distribution of water uptake from the soil profile under high soil moisture conditions was the same as the distribution of root length density. The roots in the top layer of soil played an important role in soil water uptake. When root length density was less than 0 · 8 cm cm−3, the root was the main factor limiting the complete utilization of soil water by crops. The scarcity of roots in the deep soil layers restricted the full utilization of soil water by the crops. Thus, at maturity, over 100 mm of available water remained in the root zone for the rain‐fed treatment, although the upper layers had already entered water deficit. The crop took up 80% of the total available soil water in the treatment without irrigation in a dry season. For irrigated wheat, from 40 to 50% of crop water uptake was from the stored soil water. Available stored soil water played an important role in the higher production of wheat crops in the NCP. Effective measures to increase the utilization of stored soil water could improve crop performance under conditions of limited water supply. Results showed that deep tillage to break the soil pan improved root growth in the deeper soil layers, and sowing the crop evenly also enhanced water uptake from the top soil layer to compete with soil evaporation. Copyright © 2004 John Wiley & Sons, Ltd.
Changes in components of the hydrological cycle in the Yellow River basin during the second half of the 20th century
Authors: Liu Changming , Zheng Hongxing
Published: August 2004

The Yellow River basin of China, located in the semi‐humid, semi‐arid and arid climatic zones, is now confronted with serious problems of water deficit. With regard to intensified human activities and climatic change, knowledge about changes in the regional hydrological cycle should be seen as a key requirement in searching for an adaptation strategy in water resource use. This paper attempts to detect trends associated with hydrological cycle components in the region. The hydrological cycle components are derived from monthly precipitation and runoff date, based on the schemes proposed herein. Two methods, including linear regression and Mann–Kendall, have been applied to the detection of trends in the hydrological cycle components. For the Lanzhou station, only surface runoff showed a decreasing trend. As for Huayuankou station, the results have shown that natural runoff, surface runoff and groundwater runoff all have significant decreasing trends, whereas the decreasing trend of the other components is not significant. Impacts of human activities, climatic change and annual runoff coefficient change on the hydrological cycle components are also discussed. The study suggests that increasing water resources development and utilization is the most important factor in causing the frequent drying‐up in the main course of the Yellow River. Moreover, the similarities of the trends in precipitation and natural runoff suggest a linkage between climatic change and hydrological cycle change. Copyright © 2004 John Wiley & Sons, Ltd.
Predictor relevance and extramarital affairs
Authors: Li QI , Racine Jeff
Published: August 2004

We revisit Fair's (1978) ‘theory of extramarital affairs’ using robust nonparametric methods developed for the analysis of categorical data. We find evidence suggesting that the number of years married is not a relevant predictor of the propensity to engage in extramarital affairs having controlled for other factors. This finding runs counter to the prevailing wisdom gleaned from misspecified parametric models. Copyright © 2004 John Wiley & Sons, Ltd.
Nonparametric analysis of returns to scale in the US hospital industry
Authors: Wilson Paul W. , Carey Kathleen
Published: August 2004

This paper presents new estimates of scale economies for US hospitals. We show that the common translog specification of hospital costs is a misspecification, and employ nonparametric, local linear estimation with both continuous and discrete covariates. A bootstrap method is used to provide inferences regarding ray scale economies and expansion path scale economies for a large sample of hospitals covering 1984–1996. We find evidence of changes in the structure of hospital costs over the sample period, as well as evidence of locally optimal hospital sizes. Copyright © 2004 John Wiley & Sons, Ltd.
Antibacterial activity of cloth functionalized with N‐alkylated poly(4‐vinylpyridine)
Authors: Cen L. , Neoh K. G. , Kang E. T.
Published: August 2004

A simple technique was developed to functionalize the surface of commercial cotton cloth with bactericidal polycationic groups. The cloth was first graft copolymerized with 4‐vinylpyridine (4VP) and subsequently derivatized with hexylbromide via the quaternization of the grafted pyridine groups into pyridinium groups. X‐ray photoelectron spectroscopy (XPS) was used to characterize the cotton cloth after each surface functionalization step. The pyridinium groups introduced on the substrate surface exhibited a high bacterial killing efficiency as shown by airborne and waterborne Escherichia coli (E. coli) assays, as well as a test to measure the number of viable E. coli cells in suspension upon contact with the cloth. This antibacterial property is preserved even after the cloth is subjected to prolonged weathering under UV irradiation and water spray. Wild‐type bacteria were also cultured from soil and used to verify the antibacterial potential of the functionalized cloth. The inhibition of biofilm formation on the functionalized cloth was observed even with the inoculation of high concentrations of microorganisms. The success of the present surface functionalization technique as applied to cloth is a promising development, as since cloth is a common material having wide applications. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 70–80, 2004
ABT1‐associated protein (ABTAP), a novel nuclear protein conserved from yeast to mammals, represses transcriptional activation by ABT1
Authors: Oda Tsukasa , Fukuda Aya , Hagiwara Hiroko , Masuho Yasuhiko , Muramatsu Masa‐aki , Hisatake Koji , Yamashita Takayuki
Published: August 2004

Various TATA‐binding protein (TBP)‐associated proteins are involved in the regulation of gene expression through control of basal transcription directed by RNA polymerase (Pol) II. We recently identified a novel nuclear protein, activator of basal transcription 1 (ABT1), which binds TBP and DNA, and enhances Pol II‐directed basal transcription. To better understand regulatory mechanisms for ABT1, we searched for ABT1‐binding proteins using a yeast two‐hybrid screening and isolated a cDNA clone encoding a novel protein termed ABT1‐associated protein (ABTAP). ABTAP formed a complex with ABT1 and suppressed the ABT1‐induced activation of Pol II‐directed transcription in mammalian cells. Furthermore, ABTAP directly bound to ABT1, disrupted the interaction between ABT1 and TBP, and suppressed the ABT1‐induced activation of Pol II‐directed basal transcription in vitro. These two proteins colocalized in the nucleolus and nucleoplasm and were concomitantly relocalized into discrete nuclear bodies at higher expression of ABTAP. Taken together, these results suggest that ABTAP binds and negatively regulates ABT1. The ABT1/ABTAP complex is evolutionarily conserved and may constitute a novel regulatory system for basal transcription. © 2004 Wiley‐Liss, Inc.
Differential regulation of platelet‐derived growth factor stimulated migration and proliferation in osteoblastic cells
Authors: Mehrotra Meenal , Krane Stephen M. , Walters Kristen , Pilbeam Carol
Published: August 2004

Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet‐derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3‐E1 cells and murine calvarial osteoblasts over 24–48 h. PDGF also stimulated a tenfold increase in 3H‐thymidine (3H‐TdR) incorporation in MC3T3‐E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen‐activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c‐Jun N‐terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF‐stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation. © 2004 Wiley‐Liss, Inc.
Constitutive association of cell surface CCR5 and CXCR4 in the presence of CD4
Authors: Wang Jinhai , Alvarez Raymond , Roderiquez Gregory , Guan Ennan , Norcross Michael A.
Published: August 2004

Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV‐1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF‐1α. CCR5 was resistant to ligand‐dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co‐immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV‐1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine–chemokine receptor interactions and HIV‐1 coreceptor functions. Published 2004 Wiley‐Liss, Inc.
Stretch‐induced phosphorylation of ERK1/2 depends on differentiation stage of osteoblasts
Authors: Jansen J.H.W. , Weyts F.A.A. , Westbroek I. , Jahr H. , Chiba H. , Pols H.A.P. , Verhaar J.A.N. , van Leeuwen J.P.T.M. , Weinans H.
Published: August 2004

The goal of this study was to investigate the effect of mechanical loading on osteoblasts and extracellular signal‐regulated kinase (ERK1/2) signaling in relation to osteoblast differentiation and mineralization. A human osteoblast cell line (SV‐HFO) was triggered to differentiate to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and β‐glycerophosphate. Osteoblasts were subjected to cyclic, equibiaxial stretch for 5, 15, or 60 min at different stages of differentiation (days 7, 14, and 21). Baseline (static) phosphorylated ERK1/2 and total ERK1/2 levels gradually increased during osteoblast differentiation. Cyclic stretch induced a rapid increase in ERK1/2 phosphorylation with a maximum between 5 and 15 min. Prolonged stretching for 60 min resulted in a decrease of phosphorylated ERK1/2 towards baseline level, suggesting a desensitization mechanism. The effect of stretch on ERK1/2 phosphorylation was strongest at later stages of differentiation (days 14 and 21). At day 21, the increase of ERK1/2 phosphorylation in response to stretch was significantly lower in non‐differentiating than in differentiating osteoblasts. This could not be explained by differences in cell density, but did correlate with the formation of extracellular matrix, collagen fibrils. Mineralization of the extracellular matrix did not lead to a further increase of ERK1/2 phosphorylation. In conclusion, the current study demonstrates that the extent of activation of the ERK1/2 pathway is dependent on the differentiation or functional stage of the osteoblast. The presence of an extracellular matrix, but not per se mineralization, seems to be the predominant determinant of osteoblastic response to strain. © 2004 Wiley‐Liss, Inc.
Temporal expression changes during differentiation of neural stem cells derived from mouse embryonic stem cell
Authors: Ahn Joon‐Ik , Lee Ki‐Hwan , Shin Dong‐Mi , Shim Jae‐Won , Kim Chang‐mee , Kim Hyun , Lee Sang‐Hun , Lee Yong‐Sung
Published: August 2004

Temporal analysis in gene expression during differentiation of neural stem cells (NSCs) was performed by using in‐house microarrays composed of 10,368 genes. The changes in mRNA level were measured during differentiation day 1, 2, 3, 6, 12, and 15. Out of 10,368 genes analyzed, 259 genes were up‐regulated or down‐regulated by 2‐fold or more at least at one time‐point during differentiation, and were classified into six clusters based on their expression patterns by K‐means clustering. Clusters characterized by gradual increase have large numbers of genes involved in transport and cell adhesion; those which showed gradual decrease have much of genes in nucleic acid metabolism, cell cycle, transcription factor, and RNA processing. In situ hybridization (ISH) validated microarray data and it also showed that Fox M1, cyclin D2, and CDK4 were highly expressed in CNS germinal zones and ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) was highly expressed in choroid plexus where stem/progenitor cells are possibly located. Together, this clustering analysis of expression patterns of functionally classified genes may give insight into understanding of CNS development and mechanisms of NSCs proliferation and differentiation. © 2004 Wiley‐Liss, Inc.
Paradoxical enhancement of oxidative cell injury by overexpression of heme oxygenase‐1 in an anchorage‐dependent cell ECV304
Authors: Maruhashi Keiko , Kasahara Yoshihito , Ohta Kunio , Wada Taizo , Ohta Kazuhide , Nakamura Nami , Toma Tomoko , Koizumi Shoichi , Yachie Akihiro
Published: August 2004

There has been increasing evidence suggesting the potent anti‐inflammatory roles of heme oxygenase‐1 (HO‐1) in protecting renal tubular epithelial cells, vascular endothelial cells, and circulating monocytes. Based on these findings, novel therapeutic interventions have been proposed to control the expression of endothelial HO‐1 levels to ameliorate various vascular diseases. We evaluated the effect of HO‐1 gene transfer into an anchorage‐dependent cell, ECV304. Effect of HO‐1 production on the cell injury induced by hydrogen peroxide was evaluated after hemin stimulation and after HO‐1 gene transfection. Morphological changes and the induction of various anti‐apoptotic proteins were examined at the same time. Levels of HO‐1 expression were variable in different clones of HO‐1‐transfected ECV304 cells. Among these, the clones with moderate levels of HO‐1 expression were significantly more resistant to oxidative stress. In contrast, those with the highest levels of HO‐1 exhibited paradoxically enhanced susceptibility to oxidative injury. Interestingly, the cell survival after oxidative stress was in parallel with the levels of Bcl‐2 expression and of fibronectin receptor, α5 integrin. It is suggested from these results, that excessive HO‐1 not only leads to enhanced cell injury, but also prolongs the repair process of the injured endothelial tissue. However, HO‐1 reduces the oxidative cell injury and protects the endothelial cells, if its expression is appropriately controlled. © 2004 Wiley‐Liss, Inc.
In vivo activated caspase‐3 cleaves PARP‐1 in rat liver after administration of the hepatocarcinogen N‐nitrosomorpholine (NNM) generating the 85 kDa fragment
Authors: Węsierska‐Gądek Józefa , Gueorguieva Marieta , Wojciechowski Jacek , Tudzarova‐Trajkovska Slavica
Published: August 2004

We reported previously that treatment of rats with the hepatocarcinogen N‐nitrosomorpholine (NNM) caused severe hepatotoxicity associated with apoptosis of hepatocytes beginning 12 h after administration of NNM. We observed that poly(ADP‐ribose) polymerase 1 (PARP‐1), one of the major nuclear targets for caspases, was proteolytically degraded generating primarily 64 and 54 kDa fragments. Interestingly, at 20, 30, and 40 h post‐treatment a 85 kDa cleavage product of PARP‐1 resembling that generated by caspase‐3 appeared additionally in hepatocytes. More detailed analysis performed in the present study revealed that the 85 kDa fragment of PARP‐1 was generated in the liver in 10 of 17 (60%) animals examined between 20 and 40 h after NNM administration. The caspase‐3 generated 85 kDa fragment was detected solely in hepatocytes undergoing apoptosis as evidenced by immunostaining performed with the antibody recognizing exclusively PARP‐1 cleaved at position 214/215. The appearance of the 85 kDa fragment of PARP‐1 in the liver nuclei coincided temporally with an significant increase of caspase‐3 activity in hepatocytes. In contrast, in testis samples obtained from the same animals, no changes characteristic for apoptosis such as induction of caspases activity or degradation of nuclear PARP‐1 could be detected. Our results evidence unequivocally that PARP‐1 in liver is not resistant to caspases and can be processed in vivo by activated caspase‐3 producing the p85 kDa fragment. Moreover, the caspase‐3 induced PARP‐1 fragmentation coinciding with the increase of caspase‐3 activity was detected solely in the target organ and exclusively in hepatocytes undergoing apoptosis. Considering the fact that the caspase‐3 mediated PARP‐1 cleavage occurred only in 60% of animals tested between 20 and 40 h, it becomes obvious that the cellular response in vivo to the same trigger(s) strongly varies and may depend on a variety of intrinsic factors. It remains to elucidate which additional factors may be involved in the modulation of cellular response to the strong insults thereby activating different pathways and generating distinct outcomes. © 2004 Wiley‐Liss, Inc.
The tyrosine phosphatase, OST‐PTP, is expressed in mesenchymal progenitor cells early during skeletogenesis in the mouse
Authors: Yunker Laurie A. , Undersander Anne , Lian Jane B. , Stein Gary S. , Carlson Cathy S. , Mauro Laura J.
Published: August 2004

Osteotesticular protein tyrosine phosphatase (OST‐PTP; OST), is a signaling molecule which catalyzes the removal of phosphates from tyrosine residues. It is known to be highly regulated in bone cells and has been shown to be important for the in vitro progression from a preosteoblast to a mature, mineralizing cell. However, the in vivo expression of this phosphatase during skeletogenesis has not been examined. Using Northern analysis and in situ hybridization (ISH), we have observed that this gene is strongly expressed early during the formation of the mouse skeleton. By 12.5 days post‐coitum (dpc), expression of OST mRNA transcripts increases and is localized within the mesenchyme of craniofacial bones, ribs, limbs, and Meckel's cartilage. Following initiation of chondrogenesis, OST mRNA becomes restricted to the perichondrium of all endochondral elements. With ossification, this gene is also expressed by cells, presumably osteoblasts, at the chondro‐osseous border and along cortical and trabecular bone surfaces. Unlike other bone markers examined such as Osterix and type II collagen, OST transcripts do not appear to be expressed by chondrocytes of epiphyseal cartilage or by non‐hypertrophic or hypertrophic chondrocytes. Because the temporal expression patterns of OST and Runx2 were similar suggesting a potential interrelationship in bone regulation and function, OST expression was examined in transgenic mice lacking a functional Runx2/Cbfa1 protein (Runx2/Cbfa1 delta C (ΔC)) and possessing a cartilaginous skeleton. Interestingly, the OST gene was expressed with localization similar in wild‐type, homozygous, and heterozygous embryos. These studies suggest that the expression of the OST gene may be important during skeletogenesis, potentially from commitment of mesenchymal cells to the ossification of new bones. Early in embryogenesis, regulation of OST expression may be independent of Runx2/Cbfa1. © 2004 Wiley‐Liss, Inc.
The role of stathmin in the regulation of the cell cycle
Authors: Rubin Camelia Iancu , Atweh George F.
Published: August 2004

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule‐depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell‐cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take. © 2004 Wiley‐Liss, Inc.
C‐terminal deletion mutant of MRE‐binding transcription factor‐1 inhibits MRE‐driven gene expression
Authors: Kimura Tomoki , Itoh Norio , Sone Tomomichi , Tanaka Keiichi , Isobe Masakazu
Published: August 2004

Heavy metal‐induced transcriptional activation of the genes coding for metallothionein (MT) is mediated by a cis‐acting DNA element, the metal‐responsive element (MRE). MRE‐binding transcription factor‐1 (MTF‐1) is a highly conserved heavy metal‐induced transcriptional activator. MTF‐1 also activates transcription in response to oxidative stress and regulates the expression of several cytoprotective factor genes, including MT, γ‐glutamylcysteine synthetase, and Cu/Zn‐superoxide dismutase. It is thus thought that MTF‐1 plays a role in cellular stress response. The physiological role of MTF‐1 remains unclear because of the lack of MTF‐1‐specific activators and/or inhibitors. To obtain an MTF‐1‐specific inhibitor, we constructed an MTFΔC (amino acids 1–317), a C‐terminal deletion mutant of MTF‐1. MTFΔC could bind MRE and competed with MTF‐1 for MTF–MRE complex formation. Transient expression of MTFΔC in HepG2 cells reduced MRE‐driven gene expression, demonstrating that MTFΔC is dominant to MTF‐1. HepG2 cells stably expressing MTFΔC showed increased susceptibility to the cytotoxic effects of tert‐butyl hydroperoxide (tBH). Furthermore, we constructed Ad5MTFΔC, a recombinant adenovirus that expresses MTFΔC. Infection with the virus induced MTFΔC expression and increased susceptibility to the cytotoxic effects of tBH. These results indicate that MTF‐1 participates in controlling the cellular redox state. © 2004 Wiley‐Liss, Inc.
Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4‐II‐E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644
Authors: Izumi Takako , Yamaguchi Masayoshi
Published: August 2004

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death was investigated by using the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin. The hepatoma cells (wild‐type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 12–72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 μg/ml). The number of wild‐type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 μg/ml). The effect of LPS (0.1 or 1.0 μg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 μg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca2+/calmodulin‐dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 μg/ml) for 24–72 h of wild‐type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 μg/ml)‐induced decrease in the number of wild‐type cells was significantly prevented by culture with caspase‐3 inhibitor (10−8 M). Moreover, the number of wild‐type cells was significantly decreased by culture with PD 98059 (10−6 M), dibucaine (10−6 M), or staurosporine (10−6 M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild‐type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 × 10−6 M), an agonist of Ca2+ entry in cells, caused a significant decrease in the number of wild‐type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild‐type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low‐molecular‐weight deoxyribonucleic acid (DNA) fragments of adherent wild‐type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling‐related factors. © 2004 Wiley‐Liss, Inc.
Apoptotic effect of PP2 a Src tyrosine kinase inhibitor, in murine B cell leukemia
Authors: Lee Michael , Kim Ji‐Young , Koh Woo Suk
Published: August 2004

Src is a non‐receptor protein tyrosine kinase that transduces signals regulating cell growth and differentiation. We report here that activation of signaling pathway after blockade of tyrosine phosphorylation by PP2 (4‐amino‐5‐(4‐chloro‐phenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyrimidine), a potent and selective inhibitor of the Src‐family tyrosine kinase, can lead to cell death in murine B cell leukemia, 70Z/3. Death from PP2 occurred by apoptosis as indicated by the induction of caspase activation and annexin V/propidium iodide staining. Interestingly, PP2 was found to be able to enhance the DNA binding activity of nuclear factor κB (NF‐κB) before induction of apoptosis without accompanying by increased phosphorylation of inhibitor of NF‐κB‐α (IκB‐α). Additionally, immunoblotting analysis with PP2‐treated cell extract demonstrated that, compared to other protein kinase C (PKC) isotypes, the translocation of novel PKC isotypes from the cytosol to membrane fraction was sustained for a longer time. These data suggest that the inhibition of Src‐mediated tyrosine phosphorylation by PP2 may tilt the balance between each PKC isotypes, which in turn, activate NF‐κB transcription factor, leading to apoptosis. © 2004 Wiley‐Liss, Inc.
Taming psoriatic keratinocytes—PTHs' uses go up another notch
Authors: Whitfield James F.
Published: August 2004

The native parathyroid hormone (PTH) and several of its N‐terminal adenylyl cyclase‐activating fragments and their analogs have become the star stimulators of bone growth for treating osteoporosis, accelerating fracture healing, and strengthening the anchorage of prosthetic bone implants and one of them (Lilly's Forteo™—recombinant hPTH‐(1‐34) has recently arrived in the clinic. But something entirely different has been lurking in the background—the ability of the adenylyl cyclase stimulating hPTH‐(1‐34) to calm hyperproliferating keratinocytes and reduce psoriatic lesions. By contrast PTH‐(7‐34) which cannot stimulate adenylyl cyclase actually stimulates keratinocyte proliferation. Normal keratinocytes make PTHrP after they lift off the basal lamina and have stopped cycling. But they have an unconventional PTH/PTHrP receptorr which is not coupled to adenylyl cyclase. Psoriatic keratinocytes do not make PTHrP and have only a broken‐down, proliferation‐limiting terminal differentiation‐driving Notch–Notch ligand mechanism. Putting these and other facts together produces a possible picture of an exogenously applied adenylyl cyclase‐activating PTH pinch hitting for the missing PTHrP and restoring normal keratinocyte proliferative activity epidermal structure by stimulating dermal fibroblasts which do have the conventional adenylyl cyclasse‐linked PTHR1 and in response directly or indirectly restore the overlying basal keratinocytes' Notch–Notch ligand terminal differentiation‐driving mechanism and consequently a normal epidermal structure. © 2004 Wiley‐Liss, Inc.
Elevated expression of the estrogen receptor prevents the down‐regulation of p21Waf1/Cip1 in hormone dependent breast cancer cells
Authors: Zhao Helen , Yu Jenny , Peltier Cheryl P. , Davie James R.
Published: August 2004

Expression of an estrogen receptor α (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol‐induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP‐ER) hindered entry of estrogen treated MCF‐7 cells into S phase of the cell cycle. In analyses of key cell‐cycle regulating proteins, we observed that GFP‐ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP‐ER expressing cells but not in non‐GFP‐ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down‐regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase. © 2004 Wiley‐Liss, Inc.