bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of chronic stress on hippocampal long‐term potentiation
Authors: Pavlides Constantine , Nivón Lucas G. , McEwen Bruce S.
Published: August 2002

Chronic stress causes atrophy of the apical dendrites of CA3 pyramidal neurons and deficits in spatial memory. We investigated the effects of chronic stress on hippocampal physiology and long‐term potentiation (LTP) in the CA3 and dentate gyrus (DG). Rats were subjected to chronic (21 days, 6 h/day) restraint stress and tested for LTP 48 h following the last stress episode. Control animals were briefly handled each day, similar to the experimental group but without restraint. To eliminate acute stress effects, a second control group of rats was subjected to a single acute (6 h) restraint stress and tested for LTP 48 h later. Field potential recordings were made, under chloropent anesthesia, from the stratum lucidum of CA3, with stimulation of either the mossy fiber or commissural/associational pathways, or in the DG granule‐cell layer, with stimulation of the medial perforant pathway. Chronic stress produced a suppression of LTP at 48 h compared to controls in a site‐specific manner, namely, significantly lower LTP in the medial perforant input to the DG and also in the commissural/associational input to the CA3, but not in the mossy fiber input to CA3. The animals subjected to acute stress and tested 48 h later did not show a suppression in LTP. High‐frequency stimulation (HFS) of the commissural/associational and mossy fiber inputs to CA3 produced epileptic afterdischarges in 56% of acutely stressed animals and in 29% of chronically stressed animals, whereas HFS caused afterdischarges in only 9% of nonstressed controls. No afterdischarges were seen in the medial perforant path input to DG. In order to explore the basis for these changes, we performed paired‐pulse inhibition/facilitation (PPI/F) and current‐source‐density (CSD) analysis in stressed and control animals. For PPI/F, acute stress caused an overall significant enhancement of excitation in the commissural/associational input to CA3 and medial perforant path input to DG. In contrast, chronic stress did not produce significant changes in PPI/F. The CSD analysis revealed significant chronic stress‐induced shifts in the current sources and sinks in the apical dendrites and pyramidal cell layers of the CA3 field but not in the DG. These results are consistent with the morphological findings for stress effects upon dendrites of CA3 neurons. Furthermore, they suggest that chronic stress produces changes in the input‐output relationship in the hippocampal trisynaptic circuit which could affect information flow through this structure. Hippocampus 2002;12:245–257. © 2002 Wiley‐Liss, Inc.
Factors affecting sediment trapping in vegetated filter strips: simulation study using VFSMOD
Authors: Abu‐Zreig Majed
Published: August 2002

Soil and water conservation practices have been promoted for a long time, in order to sustain agricultural activities and prevent environmental pollution. Vegetated filter strips (VFS) have been used to reduce sediment pollution into water bodies at or near the pollutant source. However, factors effecting VFS performance under natural conditions have not been well understood owing to the physical, time and financial limitations of field experiments. The use of well‐validated simulation models to understand the performance of VFS and factors affecting sediment deposition is highly justified. The objective of this research is to investigate sediment trapping in VFS and to study various factors affecting VFS performance using the simulation model VFSMOD, which was developed by researchers at University of North Carolina. Recently, VFSMOD has been validated successfully by using 21 filters with varying length, slope and vegetated cover. A wide range of five parameters was selected for the simulations, namely filter length, filter slope, manning roughness coefficient, soil type and characteristics of incoming sediment from adjacent fields. Computer simulations revealed that the length of filter is the most significant factor affecting sediment trapping in VFS. The relative increase in trapping efficiencies was not linearly related to an increase in filter length. Inflow sediment class also has a major influence on sediment trapping in VFS. The trapping efficiency of clay sediments in a 15 m length VFS was 47% compared with 92% for silt from incoming sediment. Manning roughness coefficient had a moderate effect on sediment trapping and was more significant in short filters. Land slope and soil type of VFS had a minor influence on the performance of VFS. Copyright © 2001 John Wiley & Sons, Ltd.
To go or not to go: inhibitory control in ‘hard to manage’ children
Authors: Brophy Marcia , Taylor Eric , Hughes Claire
Published: August 2002

This study is an age‐7 follow‐up of 40 children who at age 4 were identified as ‘hard to manage’ and were compared to 40 age and sex matched typically developing peers on a set of simple, manually administered executive function tasks. At age 4, the ‘hard to manage’ group showed marked deficits in inhibitory control and planning, but showed intact set‐shifting and working memory (Hughes et al., 1998). The present study assessed the continuity of executive function deficits among these ‘hard to manage’ children, using computerized tests of planning, working memory and set‐shifting (from the Cambridge Neuropsychological Test Automated Battery (CANTAB)) and a computerized Go‐No‐Go test of inhibitory control (from the Maudsley Attention and Response Suppression task battery (MARS)). In addition, real‐time observational ratings were made via a questionnaire checklist. Compared with controls, children in the ‘hard to manage’ group showed impaired inhibitory control but intact planning, working memory and set‐shifting. Observational ratings confirmed the group difference in inhibitory control, as the ‘hard to manage’ group showed significantly more perseverative errors and rule violations. These findings raise important methodological issues concerning effects of task format and highlight the importance of combining experimental and observational approaches to assessing problems in executive control. Copyright © 2002 John Wiley & Sons, Ltd.
Antiangiogenic activity of endostatin inhibits c6 glioma growth
Authors: Peroulis Irene , Jonas Nadeen , Saleh Mary
Published: August 2002

Angiogenesis is a vital component of the development and progression of many human solid tumors. Glioblastoma multiforme is one of the most highly vascularised class of solid tumors. Thus, we have investigated the potential antitumourigenic activity of endostatin, an angiogenic inhibitor, in the rat C6 glioma model. We have engineered C6 cells that endogenously express mouse endostatin in order to assess the growth of C6 tumors in vivo when endostatin is constitutively expressed. Endostatin secreted by stably transfected C6 cells is biologically active as shown by its inhibition (26%) of bFGF‐stimulated proliferation of BAECs in culture. The subcutaneous implantation of endostatin‐C6 cells in athymic (nu/nu) mice resulted in a reduced tumor growth rate (90% inhibition) compared to control cell lines throughout the duration of our experiments. Tumor inhibition was associated with a 50% reduction in the number of vessels, which were also smaller in morphology. However, endostatin‐C6 tumors were no more necrotic than control tumors. The implantation of endostatin‐C6 cells into immunocompetent Wistar rat brains also resulted in reduced tumor volumes (71% inhibition) compared to controls. Tumor cells were sparsely localised along the injection tract but had not formed discrete tumors. Despite the inhibitory response mediated by endostatin on C6 growth, complete tumor inhibition or dormancy was not observed in either the athymic or immunocompetent tumor models. These findings demonstrate that the endogenous expression of endostatin by C6 glioma cells results in a reduced tumor growth rate in vivo that is associated with an inhibition of tumor angiogenesis. Our data suggest that endostatin should be developed as an adjuvant gene therapy for the effective treatment of gliomas. © 2001 Wiley‐Liss, Inc.
Peptide‐specific CD8+ T‐cell evolution in vivo: Response to peptide vaccination with Melan‐A/MART‐1
Authors: Jäger Elke , Höhn Hauni , Necker Artje , Förster Reinhold , Karbach Julia , Freitag Kirsten , Neukirch Claudia , Castelli Chiara , Salter Russell D. , Knuth Alaxander , Maeurer Markus J.
Published: August 2002

Monitoring of CD8+ T‐cell responses in cancer patients during peptide vaccination is essential to provide useful surrogate markers and to demonstrate vaccine efficacy. We have longitudinally followed CD8+ T‐cell responses in 3 melanoma patients who were immunized with peptides derived from Melan‐A/MART‐1. Recombinant HLA‐A2 tetramers loaded with the naturally presented Melan‐A/MART‐1 nonamer peptide (AAGIGILTV) and the Melan‐A/MART‐1 analog (ELAGIGILTV) were used in combination with phenotypical analysis for different T‐cell subsets including naive T cells, effector T cells, “true memory” T cells and “memory effector” T cells, based on CD45RA/RO and CCR7‐expression. At least in a single patient, T cells binding to the AAGIGILTV epitope were detected in naive, precursor (CD45RA+/CCR7+) CD8+ T cells, and CD8+ T cells binding to the analog ELAGIGILTV peptide were identified in the terminally differentiated (CD45RA+/CCR7−) T‐cell subset. Molecular and functional analysis of tetramer‐binding T cells revealed that the T‐cell receptor (TCR) repertoire was oligo/polyclonal in AAGIGILTV‐reactive T cells, but different and restricted to a few TCR clonotypes in ELAGIGILTV‐reactive T cells prior to vaccination. The TCR repertoire reactive with Melan‐A/MART‐1 peptide epitopes was broadened during vaccination and exhibited a different profile of cytokine release after specific stimulation: tetramer‐binding T cells from 2/3 patients secreted granulocyte/macrophage colony‐stimulating factor (GM‐CSF) and interferon‐γ but not interleukin‐2 (IL‐2) in response to Melan‐A/MART‐1 peptides. In the third patient, tetramer‐binding T cells secreted IL‐2 exclusively. Our results show that T‐cell responses to peptide vaccination consist of different T‐cell subsets associated with different effector functions. Complementary analysis for TCR CDR3 and cytokine profiles may be useful to define the most effective CD8+ T‐cell population induced by peptide vaccination. © 2002 Wiley‐Liss, Inc.
Therapeutic efficacy of IL‐2‐loaded hydrogels in a mouse tumor model
Authors: De Groot Cornelis J. , Cadée Jenny A. , Koten Jan‐Willem , Hennink Wim E. , Den Otter Willem
Published: August 2002

Interleukin‐2 (IL‐2) is a highly effective anticancer drug if it is applied locally for 5 consecutive days. In most cases this requires 5 invasive treatments, which is not usually acceptable for either the patient or the clinician. For this reason we have developed dextran‐based hydrogels from which the required amount of encapsulated IL‐2 (1–4 × 106 IU of IL‐2) is gradually released during 5–10 days. Initially IL‐2‐containing macroscopic cylinder‐shaped gels (implants), and later IL‐2‐containing injectable microspheres, were developed. These preparations were characterized in vitro, and the therapeutic activity was tested in DBA/2 mice with SL2 lymphosarcoma. The therapy was given to mice with a large and extensively metastasized tumor load (at least 5% of the body weight). If 1–4 × 106 IU of IL‐2 was slowly released from the hydrogels over a period of 5–10 days, the therapeutic effects were very good and comparable to the effects of free IL‐2 injections for 5 consecutive days. In conclusion, dextran‐based hydrogels are promising systems for the controlled release of IL‐2. © 2001 Wiley‐Liss, Inc.
CDH1 mutations are present in both ductal and lobular breast cancer, but promoter allelic variants show no detectable breast cancer risk
Authors: Lei Haixin , Sjöberg‐Margolin Sara , Salahshor Sima , Werelius Barbro , Jandáková Eva , Hemminki Kari , Lindblom Annika , Vořechovský Igor
Published: August 2002

Mutations and diminished expression of the E‐cadherin gene (CDH1) have been identified in a number of epithelial malignancies. Although somatic CDH1 mutations were detected in lobular breast cancer with a frequency ranging from 10–56%, CDH1 alterations in more frequent ductal tumors appear to be rare. Here we have analyzed the coding region of CDH1 for mutations using denaturing high performance liquid chromatography and found 4 mutations in 83 ductal carcinomas (5%) and 3 mutations in 25 lobular carcinomas (12%). The germline of 13 patients with familial lobular tumors was also analyzed for mutations, but none were detected. In a case‐control study, we also tested whether a variant adenine allele in the promoter polymorphism −161C→A with a putative influence on the transcriptional activity of CDH1 in vitro confers any detectable risk of breast cancer. No significant difference in the allelic frequency between patients with breast cancer (326/1,152, 28.3%) and controls (190/696, 27.3%, p > 0.05; relative risk 1.05, 95% confidence interval 0.85–1.30) was found. A novel promoter polymorphism was identified at position −152, but the frequency of the variant cytosine allele was also similar in patients with breast cancer and controls (0.71% vs. 0.21%, p = 0.23). Transient transfection experiments using reporter constructs containing the nucleotide substitutions −161C/−152C and −161A/−152T showed only a slight decrease in the transcription activity compared to the wild‐type construct. These results do not support CDH1 as a prominent low‐penetrance cancer susceptibility gene, but indicate that CDH1 mutations contribute to the progression of both lobular and ductal tumors. © 2001 Wiley‐Liss, Inc.
Overexpression of the eukaryotic translation initiation factor 4G (eIF4G‐1) in squamous cell lung carcinoma
Authors: Bauer Christine , Brass Nicole , Diesinger Isabel , Kayser Klaus , Grässer Friedrich A. , Meese Eckart
Published: August 2002

eIF4G‐1 belongs to the family of translational initiation factors and is recognized as the central organizing protein in recruitment of mRNA during translational initiation. Previously published studies have provided some evidence that overexpression of translational factors is a general event in the process of carcinogenesis. We have characterized the expression of the eIF4G‐1 protein in 33 squamous cell carcinoma (SCC) of the lung by Western blotting. Overexpression of the eIF4G‐1 protein was detected in 61% of the tumors compared to the respective normal lung tissue. In addition, we analyzed the expression of this protein by immunohistochemistry in 138 SCC of the lung using a newly generated antibody that is specific for eIF4G‐1 as determined by Western blotting. This anti‐eIF4G‐1 antibody was suitable for the immunohistochemistry of paraffin‐embedded tissues. There is a strong cytoplasmic staining detected in the tumor areas that is consistent with the cytoplasmic localization of the translation factor eIF4G‐1. In 72% of the examined tissue sections of SCCs of the lung, we detected an overexpression of the eIF4G‐1 protein compared to the surrounding connective tissue. Two tumors that were analyzed by both methods showed an overexpression of eIF4G‐1 both with Western blot analysis and immunohistochemical staining. Overexpression of eIF4G‐1 may result in an increased amount of the translation initiation complex eIF4F, which in turn may activate the translation of the same target mRNAs as eIF4E. © 2001 Wiley‐Liss, Inc.
Expression of interleukin‐18 in human ovarian carcinoma and normal ovarian epithelium: Evidence for defective processing in tumor cells
Authors: Wang Zhao Yuan , Gaggero Alessia , Rubartelli Anna , Rosso Ombretta , Miotti Silvia , Mezzanzanica Delia , Canevari Silvana , Ferrini Silvano
Published: August 2002

Interleukin‐18 (IL‐18) is a proinflammatory monokine structurally related to IL‐1β that stimulates interferon‐γ (IFN‐γ) production. IL‐18 is synthesized as an inactive precursor, pro‐IL‐18, which is cleaved by IL‐1β‐converting enzyme (ICE)/caspase‐1 in a mature protein. In view of the proposed use of IL‐18 in cancer immuno/gene therapy, we have studied the expression of IL‐18 in tumor cells. IL‐18 mRNA was detected by reverse transcriptase polymerase chain reaction in all human ovarian carcinoma cell lines tested (9/9) and in one‐half of tumor cell populations obtained from ovarian carcinoma patients (4/8). ICE mRNA was expressed in a smaller fraction of samples (3/9 cell lines and 3/8 samples from patients). IL‐18 protein was also found in 7/13 ovarian carcinoma solid tumors by immunohistochemic analysis. In tumor cell lines we were able to detect abundant intracellular pro‐IL‐18 (24 kDa) by Western blotting, whereas the mature form of IL‐18 was undetectable, irrespective of the presence of ICE mRNA and protein. Only pro‐IL‐18 was also found in the ovarian carcinoma cell supernatants, which did not display any IL‐18 biologic activity in functional assays. Normal cultured ovarian epithelial cells revealed the presence of both IL‐18 and ICE mRNA in all samples (5/5) and IL‐18 protein was expressed by the thin epithelial cell layer surrounding normal ovary. More importantly, normal ovarian epithelial cells released low but detectable amounts of mature IL‐18 in the culture supernatant, which displayed IL‐18‐like biologic activity in functional assays. These data suggest that mature biologically active IL‐18 production is a feature of the normal ovarian surface epithelium lost during neoplastic transformation. © 2002 Wiley‐Liss, Inc.
Neuroendocrine tumor targeting: Study of novel gallium‐labeled somatostatin radiopeptides in a rat pancreatic tumor model
Authors: Froidevaux Sylvie , Eberle Alex N. , Christe Martine , Sumanovski Lazar , Heppeler Axel , Schmitt Jörg S. , Eisenwiener Klaus , Beglinger Christoph , Mäcke Helmut R.
Published: August 2002

Somatostatin analogs labeled with radionuclides are of considerable interest in the diagnosis and therapy of SSTR‐expressing tumors, such as gastroenteropancreatic, small cell lung, breast and frequently nervous system tumors. In view of the favorable physical characteristics of the Ga isotopes 67Ga and 68Ga, enabling conventional tumor scintigraphy, PET and possibly internal radiotherapy, we focused on the development of a Ga‐labeled somatostatin analog suitable for targeting SSTR‐expressing tumors. For this purpose, 3 somatostatin analogs, OC, TOC and TATE were conjugated to the metal chelator DOTA and labeled with the radiometals 111In, 90Y and 67Ga. They were then evaluated for their performance in the AR4‐2J pancreatic tumor model by testing SSTR2‐binding affinity, internalization/externalization in isolated cells and biodistribution in tumor‐bearing nude mice. Surprisingly, we found that, compared to 111In or 90Y, labeling with 67Ga considerably improved the biologic performance of the tested somatostatin analogs with respect to SSTR2 affinity and tissue distribution. 67Ga‐labeled DOTA‐somatostatin analogs were rapidly excreted from nontarget tissues, leading to excellent tumor‐to‐nontarget tissue uptake ratios. Of interest for radiotherapeutic application, [67Ga]DOTATOC was strongly internalized by AR4‐2J cells. Furthermore, our results suggest a link between the radioligand charge and its kidney retention. The excellent tumor selectivity of Ga‐DOTA somatostatin analogs together with the different applications of Ga in nuclear oncology suggests that Ga‐DOTA somatostatin analogs will become an important tool in the management of SSTR‐positive tumors. © 2002 Wiley‐Liss, Inc.
Variation in breast cancer risk of heterozygotes for ataxia‐telangiectasia according to environmental factors
Authors: Geoffroy‐Perez Béatrice , Janin Nicolas , Ossian Katia , Laugé Anthony , Stoppa‐Lyonnet Dominique , Andrieu Nadine
Published: August 2002

Ataxia‐telangiectasia (AT) is a rare autosomal recessive disorder, characterized by progressive neuronal degeneration, immunological deficiency, radio‐sensitivity and an increased risk of cancer. Although several studies have confirmed that AT heterozygosis increases the risk of breast cancer (BC), we do not know how exogenous factors affect this risk. We performed an epidemiological study on the cancer risks associated with AT heterozygosis in France and explored the variation in BC risk according to environmental factors, such as reproductive factors and exposure to ionizing radiation. Information on the amount of ionizing radiation received by an individual in their lifetime and on their reproductive life was collected from the living relatives of 34 AT children (175 female relatives). Consistent with previous reports and with our previous estimate on the entire retrospective cohort, we found that the risk of developing BC is 3.6‐fold higher among ATM heterozygous women. An increased risk was associated with an early age at menarche, a late age at first childbirth, nulliparity, premenopausal status and increasing periods of breast cell mitotic activity (BCMA) prior to the first childbirth. Age at menarche, age at 1st childbirth and BCMA seemed to have a stronger effect in ATM heterozygotes than in non‐ATM heterozygotes. However, the tests were not all statistically significant (only age at 1st childbirth). Surprisingly, the risk of BC decreased when the chest or breasts were irradiated. It is difficult to interpret the data because of the small sample size, but further investigations should provide a biological explanation for the variation in BC risk associated with exogenous factors according to ATM heterozygosis status. © 2002 Wiley‐Liss, Inc.
Ethni‐cities: residential patterns in the Northern European and Mediterranean metropolises – implications for policy design
Authors: Malheiros Jorge
Published: August 2002

This paper examines how the role of Southern Europe in international migration systems has changed, from that of a source to a destination region, and how this is reflected in the emergence of new patterns of ethnic segregation. Areas where immigrants cluster are also areas with relatively high levels of social and housing deprivation. As a result, ethnic residential segregation is in part an expression of social exclusion. In the first part of the paper, a statistical analysis of secondary data provides an overview of the levels and distribution of ethnic segregation in Greece, Italy, Portugal and Spain. The second part of the paper examines the implications of the spatial organisation of immigrant communities for the development of territorial policies, and explores this through a comparative study of Lisbon and Rotterdam. The analysis also deals with territorialised policies developed in areas where immigrants cluster. Copyright © 2002 John Wiley & Sons, Ltd.
Field‐scale modeling to explore salinity problems in irrigated agriculture1
Authors: Droogers P. , Torabi M. , Akbari M. , Pazira E.
Published: August 2002

One‐third of irrigated land is affected by salinity or is expected to become so in the near future. A rapid assessment to evaluate the effect of changes in the quality and quantity of irrigation water was tested for the Rudasht irrigation project in Iran. This was performed by using a physically based, well‐tested simulation model for crop growth, water and salt transport at field scale. Data used were from an existing dataset and missing data were obtained by converting data from the available set to the required ones. Results indicate that the current practice of 900 mm annual application rates for cotton, given the current salinity level of 4 dS m−1, is close to the optimal one. Graphs are presented to evaluate the effect of different combinations of application rates and salinity levels on yields, and the water and salt balances. It was concluded that the methodology presented here is versatile, rapid, and transferable to other conditions. Moreover, the method produces output at a high spatial and temporal resolution over a long time frame. Copyright © 2001 John Wiley & Sons, Ltd.
Predicting direction shifts on Canadian–US exchange rates with artificial neural networks
Authors: Davis Jefferson T. , Episcopos Athanasios , Wettimuny Sannaka
Published: August 2002

The paper presents a variety of neural network models applied to Canadian–US exchange rate data. Networks such as backpropagation, modular, radial basis functions, linear vector quantization, fuzzy ARTMAP, and genetic reinforcement learning are examined. The purpose is to compare the performance of these networks for predicting direction (sign change) shifts in daily returns. For this classification problem, the neural nets proved superior to the naïve model, and most of the neural nets were slightly superior to the logistic model. Using multiple previous days' returns as inputs to train and test the backpropagation and logistic models resulted in no increased classification accuracy. The models were not able to detect a systematic affect of previous days' returns up to fifteen days prior to the prediction day that would increase model performance. Copyright © 2001 John Wiley & Sons, Ltd.
Decreased serum testosterone levels in rats exposed intraperitoneally to bismuth subnitrate
Authors: Stoltenberg Meredin , Flyvbjerg Allan , Søndergaard Liselotte G , Rungby Jørgen
Published: August 2002

The consumption of bismuth is increasing and knowledge of the potential teratogenic and reproductive damage of bismuth exposure is fragmentary. In the present study we used autometallography (AMG)—the histochemical silver amplification technique—to trace bismuth in the testis and pituitary glands of Wistar rats injected intraperitoneally with bismuth subnitrate. Large amounts of bismuth AMG grains were concentrated in the lysosomes of Leydig cells, and serum testosterone levels were reduced when compared with controls. No histochemical traces of bismuth were found in the anterior lobe of the pituitary gland. Compared with their corresponding controls, neither follicle‐stimulating hormone nor luteinizing hormone were affected. The selective uptake of bismuth in Leydig cells, followed by decreased testosterone levels, emphasizes a potential hazard of bismuth‐provoked male reproductive impairment. Copyright © 2002 John Wiley & Sons, Ltd.
Viability, adhesion, and bone phenotype of osteoblast‐like cells on polyelectrolyte multilayer films
Authors: Tryoen‐Tóth Petra , Vautier Dominique , Haikel Youssef , Voegel Jean‐Claude , Schaaf Pierre , Chluba Johanna , Ogier Joëlle
Published: August 2002

The aim of this study was to develop new biocompatible coatings for bone implants by the alternating deposition of oppositely charged polyelectrolytes. Polyelectrolyte films were built up with different terminating layers on which SaOS‐2 osteoblast‐like cells and human periodontal ligament (PDL) cells were grown. The terminating layer was made of one of the following polyelectrolytes: poly(ethylene imine) (PEI), poly(sodium 4‐styrenesulfonate) (PSS), poly(allylamine hydrochloride) (PAH), poly(L‐glutamic acid) (PGA), or poly(L‐lysine) (PLL). Cell adherence, viability, stability of osteoblast phenotype, and inflammatory response were studied. Adherence and viability were good on all terminating layers except the PEI‐terminating layer, which was cytotoxic. Maintenance of osteoblast phenotype marker expression was observed on PSS‐ and PGA‐terminating films for both cell types, whereas downregulation, associated with the induction of Interleukin‐8 (IL‐8) secretion, was detected on PEI and PAH for both cell types and on PLL for PDL cells. These results suggested a good biocompatibility of PSS‐ and PGA‐ending films for PDL cells and of PSS‐, PGA‐, and PLL‐terminating films for SaOS‐2 cells. As a result, polyelectrolyte multilayer films could emerge as new alternatives for implant coatings. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 60: 657–667, 2002
Interactions of 3T3 fibroblasts and endothelial cells with defined pore features
Authors: Salem A. K. , Stevens R. , Pearson R. G. , Davies M. C. , Tendler S. J. B. , Roberts C. J. , Williams P. M. , Shakesheff K. M.
Published: August 2002

The colonization of biodegradable polymer scaffolds with cell populations has been established as the foundation for the engineering of a number of tissues, including cartilage, liver, and bone. Within these scaffolds, the cells encounter a porous environment in which they must migrate across the convoluted polymer surface to generate a homogenous cell distribution. Predicting the interactions between cells and pores is important if scaffold characteristics are to be optimized. Therefore, we investigated the behavior of two model cell types over a range of defined pore features. These pore features range from 5 to 90 μm in diameter and have been fabricated by photolithographic techniques. Quantitatively, the behavior of the cells is dependent on three factors: 1) percentage cell coverage of the surface; 2) pore size; and 3) cell type. Fibroblast cells displayed a co‐operative pattern of cell spreading in which pores with diameters greater than the cell dimensions were bridged by groups of cells using their neighbors as supports. Endothelial cells were unable to use neighbors as support structures and failed to bridge pores greater than the cell diameter. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 61: 212–217, 2002
Modulation of insulin transport in rat brain microvessel endothelial cells by an ecto‐phosphatase activity
Authors: Calhau C. , Martel F. , Pinheiro‐Silva S. , Pinheiro H. , Soares‐da‐Silva P. , Hipólito‐Reis C. , Azevedo I.
Published: August 2002

The physiological function of alkaline phosphatase (ALP) remains controversial. It was recently suggested that this membrane‐bound enzyme has a role in the modulation of transmembranar transport systems into hepatocytes and Caco‐2 cells. ALP activity expressed on the apical surface of blood‐brain barrier cells, and its relationship with 125I‐insulin internalization were investigated under physiological conditions using p‐nitrophenylphosphate (p‐NPP) as substrate. For this, an immortalized cell line of rat capillary cerebral endothelial cells (RBE4 cells) was used. ALP activity and 125I‐insulin internalization were evaluated in these cells. The results showed that RBE4 cells expressed ALP, characterized by an ecto‐oriented active site which was functional at physiological pH. Orthovanadate (100 μM), an inhibitor of phosphatase activities, decreased both RBE4‐ALP activity and 125I‐insulin internalization. In the presence of l‐arginine (1 mM) or adenosine (100 μM) RBE4‐ALP activity and 125I‐insulin, internalization were significantly reduced. However, d‐arginine (1 mM) had no significant effect. Additionally, RBE4‐ALP activity and 125I‐insulin internalization significantly increased in the presence of the bioflavonoid kaempferol (100 μM), of the phorbol ester PMA (80 nM), IBMX (1 mM), progesterone (200 μM and 100 μM), β‐estradiol (100 μM), iron (100 μM) or in the presence of all‐trans retinoic acid (RA) (10 μM). The ALP inhibitor levamisole (500 μM) was able to reduce 125I‐insulin internalization to 69.1 ± 7.1% of control. Our data showed a positive correlation between ecto‐ALP activity and 125I‐insulin incorporation (r = 0.82; P < 0.0001) in cultured rat brain endothelial cells, suggesting that insulin entry into the blood‐brain barrier may be modulated through ALP. J. Cell. Biochem. 84: 389–400, 2002. © 2001 Wiley‐Liss, Inc.
IGF‐1 induces Pin1 expression in promoting cell cycle S‐phase entry
Authors: You Han , Zheng Hongwu , Murray Steven A. , Yu Qiang , Uchida Takafumi , Fan Daiming , Xiao Zhi‐Xiong Jim
Published: August 2002

Insulin‐like growth factor I (IGF‐1) is a well‐established mitogen to many different cell types and is implicated in progression of a number of human cancers, notably breast cancer. The prolyl isomerase Pin1 plays an important role in cell cycle regulation through its specific interaction with proteins that are phosphorylated at Ser/Thr‐Pro motifs. Pin1 knockout mice appear to have relatively normal development yet the Pin1−/−mouse embryo fibroblast (MEF) cells are defective in re‐entering cell cycle in response to serum stimulation after G0 arrest. Here, we report that Pin1−/− MEF cells display a delayed cell cycle S‐phase entry in response to IGF stimulation and that IGF‐1 induces Pin1 protein expression which correlates with the induction of cyclin D1 and RB phosphorylation in human breast cancer cells. The induction of Pin1 by IGF‐1 is mediated via the phosphatidylinositol 3‐kinase as well as the MAP kinase pathways. Treatment of PI3K inhibitor LY294002 and the MAP kinase inhibitor PD098059, but not p38 inhibitor SB203580, effectively blocks IGF‐1‐induced upregulation of Pin1, cyclin D1 and RB phosphorylation. Furthermore, we found that Cyclin D1 expression and RB phosphorylation are dramatically decreased in Pin1−/− MEF cells. Reintroducing a recombinant adenovirus encoding Pin1 into Pin1−/− MEF cells restores the expression of cyclin D1 and RB phosphorylation. Thus, these data suggest that the mitogenic function of IGF‐1 is at least partially linked to the induction of Pin1, which in turn stimulates cyclin D1 expression and RB phosphorylation, therefore contributing to G0/G1‐S transition. J. Cell. Biochem. 84: 211–216, 2002. © 2001 Wiley‐Liss, Inc.
Modulation of HeLa cells spreading by the non‐receptor tyrosine kinase ACK‐2
Authors: Coon Melissa , Herrera Roman
Published: August 2002

The CDC42 regulated non‐receptor tyrosine kinase ACK‐2 has been associated with integrin signaling. In this report, the effect of ACK‐2 on the modulation of cell spreading and motility was examined. HeLa cells expressing epitope‐tagged wild type ACK‐2 showed a slower rate of spreading on fibronectin when compared with untransfected cells. An ACK‐2 protein lacking its SH3 domain was still capable of modulating HeLa cell spreading suggesting that its tyrosine kinase activity is sufficient to induce the observed phenotype. The ACK‐2 effect on the rate of cell spreading did not involve inhibition of integrin‐mediated activation of PI‐3K signaling, since it did not alter membrane translocation of a GFP‐PH‐AKT domain (AKT pleckstrin homology domain) used as a reporter for PI‐3K products induced by cell adhesion. The ACK‐2 effect appears to be upstream from the adapter protein CrkII, since co‐expression of CrkII and ACK‐2 results in a neutralization of ACK‐2 mediated effects on HeLa cell spreading. Similarly, co‐expression of p130Cas, which interacts with the adapter protein CrkII, with ACK‐2, also results in a partial reversion of the ACK‐2 effects on cell spreading. CrkII mediated reversal of the ACK‐2 induced phenotype requires the activity of the small GTPase, Rap1. Co‐expression of ACK‐2 and CrkII with a dominant negative form of Rap1 reverses the neutralization by CrkII suggesting that CrkII mediated activation of Rap1 is required. However, an active form of Rap1 is not sufficient to reverse the ACK‐2 phenotype by itself. A role for Rac1 in ACK‐2 effects was also established. An activated Rac1 protein neutralized the ACK‐2 mediated inhibition of cell spreading. A direct measurement of cell motility by either a modified Boyden chamber or wounding assay demonstrates that ACK‐2 overexpression increases the motility of the cells. These results suggest that ACK‐2 modulates HeLa cells spreading upstream of pathways regulated by CrkII and that ACK‐2 may regulate cell motility by controlling the activation of small GTPases such as Rap1 and Rac1. J. Cell. Biochem. 84: 655–665, 2002. © 2001 Wiley‐Liss, Inc.
Phytoestrogen genistein stimulates the production of osteoprotegerin by human trabecular osteoblasts
Authors: Viereck Volker , Gründker Carsten , Blaschke Sabine , Siggelkow Heide , Emons Günter , Hofbauer Lorenz C.
Published: August 2002

The anti‐resorptive effects of estrogen on bone metabolism are thought to be mediated through modulation of paracrine factors produced by osteoblastic lineage cells that act on osteoclastic lineage cells. Receptor activator of nuclear factor‐κB ligand (RANKL) is the essential factor for osteoclast formation and activation and enhances bone resorption. By contrast, osteoprotegerin (OPG), which is produced by osteoblastic lineage cells acts as a decoy receptor that neutralizes RANKL and prevents bone loss. Recently, 17β‐estradiol was found to stimulate OPG mRNA levels and protein secretion in a human osteoblastic cell line through activation of the estrogen receptor (ER)‐α. In this study, we assessed the effects of the phytoestrogen genistein on OPG mRNA steady state levels (by semiquantitative RT‐PCR and Northern analysis) and protein production (by ELISA) in primary human trabecular osteoblasts (hOB) obtained from healthy donors. Genistein increased OPG mRNA levels and protein secretion by hOB cells by up to two‐ to six‐fold in a dose‐ (P < 0.0001) and time‐dependent (P < 0.0001) fashion with a maximum effect at 10−7 M. Co‐treatment with the pure ER antagonist ICI 182,780 completely abrogated the stimulatory effects of genistein on OPG protein secretion, indicating that these effects were specific and directly mediated through the ER. Pre‐treatment with genistein partially prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. The stimulation of OPG mRNA levels by genistein was not affected by the protein synthesis inhibitor, cycloheximide and was shown to be due to enhancement of OPG gene transcription. In conclusion, our data suggest that the phytoestrogen genistein is capable of upregulating the production of OPG by human osteoblasts. Thus, dietary sources of phytoestrogens may help to prevent bone resorption and bone loss by enhanced osteoblastic production of OPG. J. Cell. Biochem. 84: 725–735, 2002. © 2002 Wiley‐Liss, Inc.
Suppression of tissue factor expression, cofactor activity, and metastatic potential of murine melanoma cells by the N‐terminal domain of adenovirus E1A 12S protein
Authors: Voigtländer Constanze , Rand Arlymae , Liu Su‐Ling , Wilson Timothy J. , Pittelkow Mark R. , Getz Michael J. , Kelm Robert J.
Published: August 2002

Tissue factor, the cellular initiator of blood coagulation, has been implicated as a determinant of metastatic potential in human melanoma cells. Here, we report that differential expression of tissue factor in murine melanoma cell lines of known metastatic behavior is mediated by AP‐1‐dependent and 12S E1A oncoprotein‐repressible gene transcription. When compared to weakly metastatic C10 cells, highly metastatic M4 cells possessed elevated levels of tissue factor cofactor activity, transfected promoter activity, and heterodimeric AP‐1 DNA‐binding complexes containing Fra‐1. Transient co‐expression of the adenovirus E1A 12S oncoprotein strongly repressed transcription of an AP‐1‐driven tissue factor reporter gene indicating the additional requirement of N‐terminal E1A‐interacting coactivators. Stable expression of E1A mutants defective in CBP/p300‐binding failed to suppress tissue factor expression and experimental metastasis by M4 cells while clones expressing wild type E1A exhibited greatly reduced tissue factor cofactor activity and metastatic potential in vivo. Overexpression of functional tissue factor in cells containing wild type E1A failed to restore the highly metastatic M4 phenotype suggesting that additional E1A‐responsive and CBP/p300‐dependent genes are required to facilitate metastasis of murine melanoma cells demonstrating high tissue factor expression and cofactor activity. J. Cell. Biochem. 85: 54–71, 2002. © 2002 Wiley‐Liss, Inc.
NERF2, a member of the Ets family of transcription factors, is increased in response to hypoxia and angiopoietin‐1: A potential mechanism for Tie2 regulation during hypoxia
Authors: Christensen Rial A. , Fujikawa Koshi , Madore Rebecca , Oettgen Peter , Varticovski Lyuba
Published: August 2002

Vascular endothelial growth factor (VEGF) and angiopoietins regulate endothelial cell survival and migration and are essential for angiogenesis. Considerable progress has been made towards understanding hypoxia‐mediated regulation of VEGF and its receptors. In contrast, little is known about the regulation of angiopoietins and their receptors in hypoxic cells. Using RT–PCR, RNAase protection assay, and Western blotting, we found that Tie1 and Tie2 mRNA and protein levels increased in response to hypoxia in human umbilical vein endothelial cells. Previously, we have shown that NERF2, a member of Ets family of transcription factors that is specifically expressed in endothelial cells, binds to the promoter region of Tie2 and transactivates Tie2 expression. In this study, we show that expression of NERF2 was increased under hypoxia and that this increase temporally correlated with the increase in Tie2 expression. Hypoxia‐induced expression of NERF2 and Tie2 was blocked by angiopoietin‐2, a competitive inhibitor of angiopoietin‐1, and by recombinant soluble extracellular domain of Tie2 but not by VEGF‐neutralizing antibodies. In addition, angiopoietin‐1 directly induced expression of NERF2 in quiescent cells. These novel findings suggest that angiopoietin‐1 regulates expression of NERF2 and its own receptor in hypoxic cells. J. Cell. Biochem. 85: 505–515, 2002. © 2002 Wiley‐Liss, Inc.
Characterization of tissue‐specific LIM domain protein (FHL1C) which is an alternatively spliced isoform of a human LIM‐only protein (FHL1)
Authors: Ng Enders Kai On , Lee Simon M. Y. , Li Hoi‐Yeung , Ngai Sai‐Ming , Tsui Stephen K. W. , Waye Mary M. Y. , Lee Cheuk‐Yu , Fung Kwok‐Pui
Published: August 2002

We have cloned and characterized another alternatively spliced isoform of the human four‐and‐a‐half LIM domain protein 1 (FHL1), designated FHL1C. FHL1C contains a single zinc finger and two tandem repeats of LIM domains at the N‐terminus followed by a putative RBP‐J binding region at the C‐terminus. FHL1C shares the same N‐terminal two‐and‐a‐half LIM domains with FHL1 but different C‐terminal protein sequences. Due to the absence of the exon 4 in FHL1C, there is a frame‐shift in the 3′ coding region. Sequence analysis indicated that FHL1C is the human homolog of murine KyoT2. The Northern blot and RT‐PCR results revealed that FHL1 is widely expressed in human tissues, including skeletal muscle and heart at a high level, albeit as a relatively low abundance transcript in brain, placenta, lung, liver, kidney, pancreas, and testis. In contrast, FHL1C is specifically expressed in testis, skeletal muscle, and heart at a relatively low level compared with FHL1. The expression of FHL1C transcripts was also seen in aorta, left atrium, left, and right ventricles of human heart at low level. Immunoblot analysis using affinity‐purified anti‐FHL1C antipeptide antibodies confirmed a 20 kDa protein of FHL1C in human skeletal muscle and heart. Unlike FHL1B, which is another FHL1 isoform recently reported by our group and localized predominantly in the nucleus [Lee et al., 1999], FHL1C is localized both in the nucleus and cytoplasm of mammalian cell. J. Cell. Biochem. 82: 1–10, 2001. © 2001 Wiley‐Liss, Inc.
Human T cell responses to endogenously presented HLA‐A*0201 restricted peptides of simian virus 40 large T antigen
Authors: Velders Markwin P. , Macedo M. Fatima , Provenzano Maurizio , Elmishad Amira G. , Holzhütter Hermann‐Georg , Carbone Michele , Kast W. Martin
Published: August 2002

Presence of the simian virus 40 (SV40) has recently been demonstrated in a relatively high percentage of human mesotheliomas and it is associated with the development of these malignancies in pleural cavities. Therefore, we have initiated a study to identify candidate peptides presented by the human HLA‐A*0201 molecule for vaccination approaches against SV40 and monitoring of SV40 directed human immune responses. Initial screening of SV40 large T (Tag) domains required for transformation of cells for HLA‐A*0201 binding motifs revealed ten possible binding peptides. Screening of these candidate peptides showed that seven of the ten peptides could bind and stabilize HLA‐A*0201 molecules. In an in vitro immunization assay the two peptides with the highest binding affinity for HLA‐A*0201, Tag aa 396–405 and aa 577–585, were tested for their ability to induce peptide specific cytotoxic T cells in two healthy donors. One donor developed cytotoxic T cells against Tag aa 396–405 and in T cell cultures of both donors Tag aa 577–585 specific T cells were initiated. The T cells against Tag aa 577–585 not only recognized and killed peptide pulsed cells, but, most importantly, SV40 transformed human mesothelial cells. This is the first demonstration of the induction of SV40 specific human cytotoxic T lymphocytes that recognize endogenously processed peptides from SV40. This peptide identification study opens the possibility to investigate immune responses against SV40 in mesothelioma patients and in individuals exposed to SV40. J. Cell. Biochem. 82: 155–162, 2001. © 2001 Wiley‐Liss, Inc.
Nerve growth factor stimulates the interaction of ZIP/p62 with atypical protein kinase C and targets endosomal localization: Evidence for regulation of nerve growth factor‐induced differentiation
Authors: Samuels Ivy S. , Seibenhener M. Lamar , Neidigh Kimberly B.W. , Wooten Marie W.
Published: August 2002

Atypical protein kinase Cs zeta and lambda/iota play a functional role in the regulation of NGF‐induced differentiation and survival of pheochromocytoma, PC12 cells [Coleman and Wooten, 1994; Wooten et al., 1999]. Here we demonstrate an NGF‐dependent interaction of aPKC with its binding protein, ZIP/p62. Although, ZIP/p62 was not a PKC‐ι substrate, the formation of a ZIP/p62‐aPKC complex in PC12 cells by NGF occurred post activation of PKC‐ι and was regulated by the tyrosine phosphorylation state of aPKC. Furthermore, NGF‐dependent localization of ZIP/p62 was observed within vesicular structures, identified as late endosomes by colocalization with a Rab7 antibody. Both ZIP/p62 as well as PKC‐ι colocalized with Rab7 upon NGF stimulation. Inhibition of the tyrosine phosphorylation state of PKC‐ι did not prevent movement of ZIP/p62 to the endosomal compartment. These observations indicate that the subcellular localization of ZIP/p62 does not depend entirely upon activation of aPKC itself. Of functional importance, transfection of an antisense p62 construct into PC12 cells significantly diminished NGF‐induced neurite outgrowth. Collectively, these findings demonstrate that ZIP/p62 acts as a shuttling protein involved in routing activated aPKC to an endosomal compartment and is required for mediating NGF's biological properties. J. Cell. Biochem. 82:452–466, 2001. © 2001 Wiley‐Liss, Inc.
Semiglobal simplex optimization and its application to determining the preferred solvation sites of proteins
Authors: Dennis S. , Vajda S.
Published: August 2002

The classical simplex method is extended into the Semiglobal Simplex (SGS) algorithm. Although SGS does not guarantee finding the global minimum, it affords a much more thorough exploration of the local minima than any traditional minimization method. The basic idea of SGS is to perform a local minimization in each step of the simplex algorithm, and thus, similarly to the Convex Global Underestimator (CGU) method, the search is carried out on a surface spanned by local minima. The SGS and CGU methods are compared by minimizing a set of test functions of increasing complexity, each with a known global minimum and many local minima. Although CGU delivers substantially better success rates in simple problems, the two methods become comparable as the complexity of the problems increases. Because SGS is generally faster than CGU, it is the method of choice for solving optimization problems in which function evaluation is computationally inexpensive and the search region is large. The extreme simplicity of the method is also a factor. The SGS method is applied here to the problem of finding the most preferred (i.e., minimum free energy) solvation sites on a streptavidin monomer. It is shown that the SGS method locates the same lowest free energy positions as an exhaustive multistart Simplex search of the protein surface, with less than one‐tenth the number of minizations. The combination of the two methods, i.e., multistart simplex and SGS, provides a reliable procedure for predicting all potential solvation sites of a protein. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 319–334, 2002