bluuebunny/crossref_metadata_2025 huggingface dataset:
A general class of association tests for family‐based data using weight functions
Authors: Liu Xin , Gordon Derek
Published: March 2003

Based on the symmetry of transmitted/nontransmitted alleles from heterozygous parents under the null hypothesis of no association, the work proposed here establishes a general statistical framework for constructing association tests with data from nuclear families with multiple affected children. A class of association tests is proposed for both diallelic and multiallelic markers. The proposed test statistics reduce to the transmission disequilibrium test for trios, to Tsu by Martin et al. ([1997] Am. J. Hum. Genet. 61:439–448) for affected sib pairs, and to the pedigree disequilibrium test by Martin et al. ([2000] Am. J. Hum. Genet. 67:146–154); [2001] Am. J. Hum. Genet. 68:1065–1067) when using affected sibships only. The association test used in simulation and for real data (sitosterolemia) is the one which has the best overall power in detecting association. This association test is generally more powerful than the association tests proposed by Martin et al. ([2000] Am. J. Hum. Genet. 67:146–154); [2001] Am. J. Hum. Genet. 68:1065–1067) when using only affected sibships. For the sitosterolemia data set, the association test has its most significant result (P‐value=0.0012) for the marker locus on the same bacterial artificial chromosome as the disease locus. Genet Epidemiol 24:208–219, 2003. © 2003 Wiley‐Liss, Inc.
Kin‐cohort evaluation of relative risks of genetic variants
Authors: Saunders Catherine L. , Begg Colin B.
Published: March 2003

The kin‐cohort study, an embedded retrospective cohort of first‐degree relatives of probands, has been used frequently in conjunction with case‐control studies to evaluate penetrance of high‐risk genotypes. However, in principle, the kin‐cohort approach can be used in conjunction with a case‐control study in which family history data are carefully recorded to examine and validate the observed association of any candidate genotype with disease. Its utility for this purpose is governed by the relative efficiency of the kin‐cohort analysis to test hypotheses about the relevant genotype‐disease odds ratios, which in turn depends strongly on various factors, notably the magnitude of the disease incidence rate and the prevalence of the aberrant genotype. In this study, we compare the efficiencies of the two analytic approaches, case‐control vs. kin‐cohort, and illustrate the results in the context of cancer epidemiology. The kin‐cohort analysis can provide substantial, important supplementary information for relatively common cancers such as breast, lung, colorectal, and prostate, and is also relatively efficient in general for the evaluation of the stronger and more rare genetic risk factors. Genet Epidemiol 24:220–229, 2003. © 2003 Wiley‐Liss, Inc.
Microglia activation influences dye coupling and Cx43 expression of the astrocytic network
Authors: Faustmann Pedro M. , Haase Claus G. , Romberg Sabine , Hinkerohe Daniel , Szlachta Dominika , Smikalla Dirk , Krause Dorothee , Dermietzel Rolf
Published: March 2003

Under inflammatory conditions, activated microglia are capable of producing proinflammatory cytokines that are reported to influence cell‐to‐cell communication. The present study was performed to evaluate the influence of microglial activation on the coupling efficiency of the astroglial network. Primary astrocyte cultures of newborn rats were cocultured with either 5% (M5) or 30% (M30) microglia. Microglial activation (rounded phagocytotic phenotype) was investigated using the monoclonal anti‐ED1 antibody, and immunofluorescence with a polyclonal anti‐Cx43 antibody was used to study astroglial Cx43 expression and distribution. Functional coupling of astrocytes was evaluated by monitoring the transfer of microinjected Lucifer yellow into neighboring cells. The data obtained can be summarized as follows: astroglia/M30 cocultures contained significantly fewer resting microglia and significantly more activated microglia than the M5 cocultures; significantly reduced astroglial Cx43 staining was found in M30 cocultures concurrently with a reduced number of dye coupled astrocytes; and the positive correlation of percent activated microglia with reduced astroglial Cx43 expression was highly significant, indicating that the degree of intercellular communication in the astroglial network may be modulated by the activation of microglia under in vitro conditions. GLIA 42:101–108, 2003. © 2003 Wiley‐Liss, Inc.
Pharmacological characterization of P2Y receptor subtypes on isolated tiger salamander Müller cells
Authors: Reifel Saltzberg Jenny M. , Garvey Katherine A. , Keirstead Susan A.
Published: March 2003

Müller cells express a variety of neurotransmitter receptors that permit them to “sense” the extracellular environment within the retina. We have used a battery of agonists and antagonists to characterize the purinergic receptor subtypes expressed on isolated tiger salamander Müller cells. Changes in intracellular calcium ion concentration ([Ca2+]i) in Müller cells were measured using the Ca2+ indicator dye Fura‐2 and digital imaging microscopy. ATP, 2‐methylthio‐ATP, 2‐methylthio‐ADP, ADP, UTP, UDP, deoxyATP, and 3′‐O‐(4‐benzoyl)benzoyl ATP evoked increases in [Ca2+]i in both the presence and absence of extracellular Ca2+. Therefore, the increases we observed were likely due to intracellular Ca2+ release mediated by G‐protein‐coupled P2Y receptor activation, rather than Ca2+ influx via P2X receptor channels. The P2Y1 receptor agonists 2‐methylthio‐ATP, 2‐methylthio‐ADP, and ADP evoked increases in [Ca2+]i that were inhibited by the P2Y1 receptor antagonists adenosine 3′‐phosphate 5′‐phosphosulfate and 2′‐deoxy‐N6‐methyleneadenosine‐3′,5′‐bisphosphate. Responses to ADP were not completely inhibited by the P2Y1 receptor antagonists. The residual response to ADP could be mediated by P2Y13 receptors. UTP evoked an increase in [Ca2+]i that was partially inhibited by suramin, suggesting that Müller cells express P2Y2 and P2Y4 receptors. The P2Y6 receptor agonist UDP, and the P2Y11 receptor agonists deoxyATP, and 3′‐O‐(4‐benzoyl)benzoyl ATP, evoked increases in [Ca2+]i in Müller cells. We conclude that isolated tiger salamander Müller cells express P2Y1, P2Y2, P2Y6, P2Y11, and possibly P2Y4 and P2Y13 receptors. Therefore, the physiological release of ATP, ADP, UTP, and UDP and/or their accumulation in the retina under pathological conditions could stimulate increases in [Ca2+]i in Müller cells. GLIA 42:149–159, 2003. © 2003 Wiley‐Liss, Inc.
Differentiation of radial glia‐like cells from embryonic stem cells
Authors: Liour Sean S. , Yu Robert K.
Published: March 2003

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia‐like cells can be generated from ES/EC cell lines. These ES/EC cell‐derived radial glia‐like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia‐like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia‐like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function. GLIA 42:109–117, 2003. © 2003 Wiley‐Liss, Inc.
Microfilament and microtubule organization and dynamics in process extension by central glia‐4 oligodendrocytes: Evidence for a microtubule organizing center
Authors: Rumsby Martin , Afsari Farinaz , Stark Meg , Hughson Elaine
Published: March 2003

Microfilaments in freshly adhering CG‐4 cells and differentiated CG‐4 oligodendrocytes are concentrated at the tips and edges of rapidly forming processes while microtubules are concentrated in new processes and extend from a concentrated spot of α‐tubulin staining in the cell body to the cell periphery. In motile bipolar CG‐4 cells, microfilaments are heavily concentrated at the flattened end of one process and along the rim of processes and the cell body: microtubules are concentrated along main processes and splay out into process tips and the cell body. In differentiated CG‐4 oligodendrocytes, microfilaments are concentrated at the many process tips, in filopodia and in fine processes, but are not obvious in main processes where separate bundles of microtubules, which diverge at process branch points, are concentrated. γ‐tubulin, involved in microtubule nucleation, is concentrated at a small discrete area in the cell body, indicative of a microtubule organizing center. Polymerization of both actin and tubulin is required for initial process elaboration. Depolymerization of microtubules, but not of microfilaments, causes complete retraction of bipolar CG‐4 cell processes. This process retraction does not occur if microfilaments are depolymerized first, indicating that process extension/retraction in motile bipolar CG‐4 cells may occur by a balance of motor protein‐driven forces as suggested for growth cone motility. Cytoskeleton organization in CG‐4 cells is very similar to that reported for oligodendrocytes. CG‐4 cells are thus a useful model for investigating the signals and mechanisms regulating oligodendrocyte process dynamics. GLIA 42:118–129, 2003. © 2003 Wiley‐Liss, Inc.
Does it matter who you are or what you gain? an experimental study of preferences for resource allocation
Authors: Schwappach David L.B.
Published: March 2003

Using an experimental conjoint‐analysis like approach, preferences for resource allocation were studied. An interactive survey was developed which was published in the World Wide Web. A convenience sample of undergraduate students participated in the study. Subjects were confronted with nine pairwise scenarios describing hypothetical patient groups in need of life‐saving treatments. The patient groups presented differed in terms of their health‐related lifestyle, socioeconomic status, age, life expectancy, quality of life after treatment and whether they had received extensive medical care in the past. Participants were asked to allocate a finite budget to each patient group. All attributes used in this study significantly influenced respondents' preferences on how to allocate the budget between patient groups. The general importance of attributes used in the QALY approach is supported by this study with quality of life being a central criterion. The distributional patterns observed were, however, different from those expected when rigorously adhering to the QALY framework: In only a very small fraction of allocations subjects distributed the entire budget strictly on the patient group expecting the highest QALY gain. The vast majority of responders was willing to trade efficiency for a more equal distribution of resources. The approach dsecribed can be used to analyze the importance people place on different attributes in resource allocation decisions and to study preferences for the final distribution of resources. Copyright © 2002 John Wiley & Sons, Ltd.
Health status and heterogeneity of cost‐sharing responsiveness: how do sick people respond to cost‐sharing?
Authors: Remler Dahlia K. , Atherly Adam J.
Published: March 2003

This paper examines whether the responsiveness of health care utilization to cost‐sharing varies by health status and the implications of such heterogeneity. First, we show theoretically that if health care utilization of those in poor health is less responsive to cost sharing, this, combined with the skewness of health expenditures in health status, leads to overestimates of the effect of cost sharing. This bias is exacerbated when elasticities are generalized to populations with greater expenditure skewness. Second, we show empirically that cost‐sharing responsiveness does differ by health status using data from the Medicare Current Beneficiary Survey. Medicare beneficiaries are stratified into health status groups based on activity of daily living (ADL) impairments and self‐reported health status. Separately, for each of the health status groups, we estimate the effect of Medigap insurance on Part B utilization using a two‐part expenditure model. We find that the change in expenditures associated with Medigap is smaller for those in poorer health. For example, when stratified using ADLs, Medigap insurance increases expenditures for ‘healthy’ groups by 36.4%, while the increase for the ‘sick’ group is 12.7%. Results are qualitatively the same for different forms of supplemental insurance and different methods of health status stratification. We develop a test to demonstrate that adjusting our results for selection bias would result in estimates of greater heterogeneity. Our results imply that a lowerbound estimate of the bias from neglecting heterogeneity is about 2–7%. Copyright © 2002 John Wiley & Sons, Ltd.
The changing public/private mix in dentistry in the UK–a supply‐side perspective
Authors: Lynch Mauricea , Calnan Michael
Published: March 2003

This paper examines the factors beyond NHS dentists' remuneration which may explain the variations in the public/private mix in general dental practitioners' workload in the UK. Given that NHS dentistry is subject to a fixed price system, the study focused mainly on non‐income supply‐side factors. Using data from a postal survey of a national random sample of dentists practising in the NHS in England in 1997, the study found that the strategies adopted by dentists in the management of NHS lists and the evidence that dentists spent significantly more time in private consultations compared with NHS consultations support concerns over equity of access to dental care and the quality of NHS dentistry. Dentists' attitudes to NHS and private dentistry revealed considerable ambiguity towards the NHS. While, on the one hand, the lack of sufficient demand for private dentistry emerged as a strong reason for remaining in the NHS, on the other, there was evidence that equity in access to dental treatment is still seen as an important principle. The implications of these findings in the context of recent discussion of the future of NHS dentistry are considered. Copyright © 2002 John Wiley & Sons, Ltd.
Design techniques for stated preference methods in health economics
Authors: Carlsson Fredrik , Martinsson Peter
Published: March 2003

This paper discusses different design techniques for stated preference surveys in health economic applications. In particular, we focus on different design techniques, i.e. how to combine the attribute levels into alternatives and choice sets, for choice experiments. Design is a vital issue in choice experiments since the combination of alternatives in the choice sets will determine the degree of precision obtainable from the estimates and welfare measures. In this paper we compare orthogonal, cyclical and D‐optimal designs, where the latter allows expectations about the true parameters to be included when creating the design. Moreover, we discuss how to obtain prior information on the parameters and how to conduct a sequential design procedure during the actual experiment in order to improve the precision in the estimates. The designs are evaluated according to their ability to predict the true marginal willingness to pay under different specifications of the utility function in Monte Carlo simulations. Our results suggest that the designs produce unbiased estimations, but orthogonal designs result in larger mean square error in comparison to D‐optimal designs. This result is expected when using correct priors on the parameters in D‐optimal designs. However, the simulations show that welfare measures are not very sensitive if the choice sets are generated from a D‐optimal design with biased priors. Copyright © 2002 John Wiley & Sons, Ltd.
Revisiting the axiom of completeness in health care
Authors: Ryan Mandy , Miguel Fernando San
Published: March 2003

Experiments concerned with modelling individual preferences are based on the assumption of completeness i.e. it is assumed that individuals have well‐defined preferences for any choice they are presented with. However, this may not be the case for goods such as health care, where individuals are not used to making choices. If this assumption is violated, the large body of experimental economic literature eliciting patient preferences in health care may be challenged. This paper reports the results of a discrete choice experiment carried out to examine the assumption of complete preferences within health care. The tests carried out are based on the comparison of preferences for three different goods for which different levels of formed preferences are expected: a supermarket; dentist consultation and bowel cancer screening. The results do not provide sufficient evidence to support this hypothesis. However, further research is required before these results are generalised. Copyright © 2002 John Wiley & Sons, Ltd.
Measuring the value of time for methadone maintenance clients: willingness to pay, willingness to accept, and the wage rate
Authors: Borisova Natalia N. , Goodman Allen C.
Published: March 2003

Three measures of the value of time – willingness to pay (WTP) for a reduction in travel time, willingness to accept (WTA) a monetary compensation to forgo it, and the wage rate – are evaluated and compared. WTP and WTA were estimated from the two‐part regressions of time price, using contingent valuation methods with primary survey data. Systematic differences are found in comparisons of WTP and WTA with the wage rate. Copyright © 2002 John Wiley & Sons, Ltd.
Analysis of an expanded width of albumin fraction by cellulose acetate membrane electrophoresis in IgA nephropathy urine before treatment
Authors: Machii Ryoko , Matsuda Kazuyuki , Hiratsuka Nobuo , Sugimoto Kayo , Hotta Osamu , Itoh Yoshihisa , Yoshida Hiroshi , Shiba Kiyoko
Published: March 2003

Cellulose acetate membrane electrophoresis with colloidal silver stain re‐vealed that the width of the albumin fraction in IgA nephropathy (IgAN) urine before treatment was significantly expanded. This phenomenon was not shown in IgAN urine after treatment or in non‐IgAN urine. There was a reverse correlation between the width of the albumin fraction and the albumin con‐centration in IgAN urine. By immuno‐fixation, Tamm‐Horsfall protein (THP) was located in the same position as the albumin band in IgAN urine before treatment; however, in the urine of a healthy subject it was located in the same position as α1‐globulin. By ELISA, the THP‐albumin complex concentration in IgAN urine before treatment was significantly higher than in the other two diseases. The width of the albumin fraction and the sodium ion concentra‐tion of the urine were significantly correlated. The THP/albumin ratio in IgAN urine before treatment was significantly higher than in the other two groups. This suggests that the characteristic expanded width of albumin found by immunofixation indicates a THP‐albumin complex, and that the sodium concentration of urine is involved in the formation of this complex. J. Clin. Lab. Anal. 17:37–43, 2003. © 2003 Wiley‐Liss, Inc.
Cellulose acetate membrane electrophoresis in the analysis of urinary proteins in patients with tubulointerstitial nephritis
Authors: Kubota Ryo , Machii Ryoko , Hiratsuka Nobuo , Hotta Osamu , Itoh Yoshihisa , Kobayashi Shizuko , Shiba Kiyoko
Published: March 2003

Urinary proteins from 14 patients with tubulointerstitial nephritis were analyzed by cellulose acetate membrane electrophoresis. Urinary total protein concentrations were measured, and urinary 15 proteins (prealbumin, albumin, α1‐microglobulin, α1‐antitrypsin, α2‐macroglobulin, haptoglobin, retinol binding protein, transferrin, β2‐microglobulin, IgA, IgG, κ‐ and λ‐light chains, cystatin C, and lysozyme) were identified by the use of a rapid and highly sensitive colloidal silver staining reagent suited for use with cellulose acetate membranes, as reported previously by Matsuda et al. (J Clin Lab Anal 15:171–174, 2001; Clin Chem47:763–766, 2001) and Hiratsuka et al. (J Clin Lab Anal 10:403–406, 1996). We also analyzed urinary total protein concentration and urinary protein fractions according to the presence of acute or nonacute interstitial nephritis. In addition, the relationship between urinary protein fraction and complications of interstitial nephritis was analyzed. The goal of this work was to find a useful index for the diagnosis of tubulointerstitial nephritis. J. Clin. Lab. Anal. 17:44–51, 2003. © 2003 Wiley‐Liss, Inc.
TAP1 gene AccI polymorphism is associated with atopic bronchial asthma
Authors: Hang Liang‐Wen , Hsia Te‐Chun , Chen Wen‐Chi , Chen Huey‐Yi , Tsai Fuu‐Jen
Published: March 2003

Asthma is a hyperresponsive airway disease that may involve inflammation responses. A transporter associated with the antigen processing 1 gene (TAP1) is involved in antigen processing, and is therefore considered to play a role in the pathogenesis of bronchial asthma. The aim of this study was to test whether the polymorphisms of the TAP1 gene are a genetic marker for susceptibility to bronchial asthma. A normal control group comprised of 43 healthy people, and 116 patients with allergic asthma were examined in this study. The polymorphism was detected by polymerase chain reaction (PCR)‐based restriction analysis. Associations between atopic bronchial asthma and TAP1 polymorphisms were evaluated. The results revealed no significant differences between normal individuals and asthmatics in regard to the TAP1 gene DpnII polymorphism (P=0.752). However, there was a significant difference between the control and asthma groups as regards the TAP1 gene AccI polymorphism (P=0.020). The odds ratio (OR) of GG homozygotes of the TAP1 AccI polymorphism was 229.8 compared with the AA homozygote group. The results show that the AccI polymorphism may be an indicator for atopic bronchial asthma. J. Clin. Lab. Anal. 17:57–60, 2003. © 2003 Wiley‐Liss, Inc.
Serum microminerals and the indices of lipid metabolism in an apparently healthy population
Authors: Abiaka Clifford , Olusi Samuel , Al‐Awadhi Adel
Published: March 2003

Serum copper and zinc concentrations were measured in 560 apparently healthy Kuwaitis (238 males and 322 females) aged 15–80 years to assess micromineral effect on the indices of lipid metabolism. Following the recommended guidelines of the European Atherosclerosis Society (EAS) and the National Cholesterol Education Program Expert Panel (NCEPEP), the incidence of dyslipidemia was assessed from enzymatic assay data of triglycerides (TG), total cholesterol (TC), high‐density lipoprotein cholesterol (HDL‐C) and low‐density lipoprotein cholesterol (LDL‐C) concentrations. Males had significantly lower TC (P=0.029) and HDL‐C (P<0.0001) levels than females, while TG were significantly (P=0.023) lower in females. The prevalence of hypercholesterolemia, hypertriglyceridemia, elevated LDL‐C, and low HDL‐C levels were 35, 30, 22, and 13%, respectively. Copper did not correlate with zinc (r = –0.067, P = 0.135) but was positively associated with TC (r=0.196, P<0.0001), LDL‐C (r=0.134, P = 0.003), TG (r = 0.092, P=0.039), and age (r=0.281, P<0.0001). It is concluded that unlike in animal studies, copper excess in humans is associated with hyperlipidemia and therefore will predispose to atherosclerosis. J. Clin. Lab. Anal. 17:61–65, 2003. © 2003 Wiley‐Liss, Inc.
Reticulocyte parameters and hemoglobin F production in sickle cell disease patients undergoing hydroxyurea therapy
Authors: Borba R. , Lima C.S.P. , Grotto H.Z.W.
Published: March 2003

Hemoglobin F (HbF) is an effective inhibitor of HbS polymerization. Hydroxyurea (HU) is used to increase HbF synthesis and improve the clinical course of sickle cell disease (SCD) patients. We studied a series of laboratory parameters concerning HbF production and reticulocyte response, and compared data between two groups: 1) 13 SCD patients treated with HU, and 2) 33 untreated SCD patients. Higher values of Hb concentration, mean cell volume (MCV), mean cell hemoglobin (MCH), mean reticulocyte volume (MRV), HbF concentration, percentage of F‐cells, and amount of HbF/F‐cells were observed in the treated group of patients. There was no correlation between Hb and HbF elevations. The reticulocyte count, immature reticulocyte count, mean fluorescence index (MFI), and neutrophil count were significantly lower in treated patients. Taken together, these findings suggest that a decreased hemolytic process occurred in patients undergoing HU treatment. There was a significant correlation between MCV and HbF, between MRV and HbF, and between MRV and F‐cell in patients taking HU. These data indicate that macroreticulocytes correspond to F‐reticulocytes, and that an increase in MRV in SCD patients using HU may be an indirect signal of F‐cell production. The concentration of HbF/F‐cells was higher in patients treated with HU, but this increase apparently was independent of F‐cell production. Reticulocyte (RTC) parameters, as assessed by hematological analyzers, may be useful for following erythropoietic changes in patients receiving HU, and can indirectly indicate HbF and F‐cell production induced by HU therapy. J. Clin. Lab. Anal. 17:66–72, 2003. © 2003 Wiley‐Liss, Inc.
Voltage‐dependent K+ channel β subunits in muscle: Differential regulation during postnatal development and myogenesis
Authors: Grande Maribel , Suàrez Elisabeth , Vicente Rubén , Cantó Carles , Coma Mireia , Tamkun Michael M. , Zorzano Antonio , Gumà Anna , Felipe Antonio
Published: March 2003

Voltage‐dependent potassium channels contribute to the electrical properties of nerve and muscle by affecting action potential shape and duration. The complexity of the currents generated is further enhanced by the presence of accessory β subunits. Here we report that while all Kvβ mRNA isoforms are present in rat brain, muscle tissues express only Kvβ1 (Kvβ1.1–Kvβ1.3) and Kvβ2, but not Kvβ3. Kvβ subunits were close regulated through post‐natal development in brain and striated muscle, as well as during myogenesis in the rat skeletal muscle cell line L6E9. While the alternatively spliced Kvβ mRNA products from Kvβ1 gene were differentially expressed, Kvβ2.1 was associated with myogenesis. These results show that Kvβ genes are strongly regulated in muscle and suggest a physiological role for voltage‐gated K+ channels during development and myotube formation. © 2003 Wiley‐Liss, Inc.
Involvement of S6 kinase and p38 mitogen activated protein kinase pathways in strain‐induced alignment and proliferation of bovine aortic smooth muscle cells
Authors: Li Wei , Chen Quanhai , Mills Ira , Sumpio Bauer E.
Published: March 2003

Bovine aortic smooth muscle cell (SMC) phenotype can be altered by physical forces. This has been demonstrated by cyclic strain‐induced changes in proliferation and alignment. However, the intracellular coupling pathways remain ill defined. In the present study, we examined whether the p38 and S6 kinase pathway were involved in the mitogenic and morphological changes seen in SMCs exposed to cyclic strain. We seeded bovine aortic SMCs on silastic membranes that were deformed with 150‐mmHg vacuum. Cyclic strain induced both alignment and proliferation of SMCs. SB202190, a specific inhibitor of p38, hindered SMC alignment, but not proliferation. Rapamycin, a specific inhibitor of the mTOR‐S6 kinase pathway, attenuated strain‐induced proliferation, but not alignment. Peak activation of p38 and S6 kinase was 351 ± 76.9% at 5 min and 363 ± 56.2% at 60 min compared with static control, respectively (P < 0.05). The results suggest that strain‐induced SMC alignment is dependent on activation of p38, but not S6 kinase. Strain induced SMC proliferation is S6 kinase, but not p38 activation, dependent. © 2003 Wiley‐Liss, Inc.
Expression and tyrosine phosphorylation of Cbl regulates macrophage chemokinetic and chemotactic movement
Authors: Caveggion Elena , Continolo Silvia , Pixley Fiona J. , Stanley E. Richard , Bowtell David D.L. , Lowell Clifford A. , Berton Giorgio
Published: March 2003

Primary macrophages isolated from hck−/−fgr−/− mice display altered morphology and F‐actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck−/−fgr−/− defects was tested. Although PMA‐treated wild‐type and hck−/−fgr−/− macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck−/−fgr−/− macrophage migration defect. Instead, both PMA‐treated wild type and hck−/−fgr−/− macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c‐cbl−/− macrophages displayed the same impairment of motility as hck−/−fgr−/− macrophages and a similar morphology with less polarization and more dorsal ruffling than wild‐type macrophages. As Hck and Fgr expression and activity were not decreased in c‐cbl−/− macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase‐regulated macrophage motility pathway. © 2003 Wiley‐Liss, Inc.
Heregulin and retinoids synergistically induce branching morphogenesis of breast cancer cells cultivated in 3D collagen gels
Authors: Offterdinger Martin , Schneider Sonja M. , Grunt Thomas W.
Published: March 2003

C‐erbB and retinoid receptor signaling control mammary epithelial cell proliferation, differentiation, and morphology. Here, we examined the morphogenetic activities of c‐erbB specific ligands such as heregulin and of retinoids on non‐malignant (primary, MTSV1‐7) and malignant (T47D, SKBR‐3) human mammary epithelial cells (HMEC) cultivated in 3D collagen type I gels. These cells are positive for both c‐erbB and retinoid receptors. Non‐malignant primary HMEC spontaneously formed branched structures in collagen, whereas SV40 large T antigen‐immortalized non‐tumorigenic MTSV1‐7 spontaneously formed balls and required heregulin or retinoid X receptor α‐selective retinoid Ro 25‐7386 for branching, which was further stimulated by combination of both types of agents. In malignant cells, heregulin alone induced ball formation and cooperated either with Ro 25‐7386 (T47D) or with retinoic acid receptor α‐selective AM580 (SKBR‐3) for branching morphogenesis, which was accompanied by changes in the subcellular distribution of α2β1‐integrin and E‐cadherin, and by down‐regulation of c‐erbB‐2, ‐3, or ‐4. Heregulin and/or retinoids correspondingly increased the integrin‐dependent adhesion of malignant cells to type I collagen. Our data demonstrate cooperative signaling of c‐erbB and retinoid receptor pathways at the levels of morphogenesis and immunophenotypic differentiation. © 2003 Wiley‐Liss, Inc.
Inhibition of protein kinase C decreases prostaglandin‐induced breakdown of the blood‐retinal barrier
Authors: Saishin Yoshitsugu , Saishin Yumiko , Takahashi Kyoichi , Melia Michele , Vinores Stanley A. , Campochiaro Peter A.
Published: March 2003

Breakdown of the blood‐retinal barrier (BRB) occurs in several retinal diseases and is a major cause of visual loss. Vascular endothelial growth factor (VEGF) has been implicated as a cause of BRB breakdown in diabetic retinopathy and other ischemic retinopathies, and there is evidence to suggest that other vasopermeability factors may act indirectly through VEGF. In this study, we investigated the effect of several receptor kinase inhibitors on BRB breakdown resulting from VEGF, tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), insulin‐like growth factor‐1 (IGF‐1), prostaglandin E1 (PGE1), or PGE2. Inhibitors of VEGF receptor kinase, including PKC412, PTK787, and SU1498, decreased VEGF‐induced breakdown of the BRB. None of the inhibitors blocked leakage caused by TNF‐α, IL‐1β, or IGF‐1 and only PKC412, an inhibitor of protein kinase C (PKC) as well as VEGF and platelet‐derived growth factor (PDGF) receptor kinases, decreased leakage caused by prostaglandins. Since the other inhibitors of VEGF and/or PDGF receptor kinases that do not also inhibit PKC had no effect on prostaglandin‐induced breakdown of the BRB, these data implicate PKC in retinal vascular leakage caused by prostaglandins. PKC412 may be useful for treatment of post‐operative and inflammatory macular edema, in which prostaglandins play a role, as well as macular edema associated with ischemic retinopathies. © 2003 Wiley‐Liss, Inc.
Activation of P2Y2 receptor induces c‐FOS protein through a pathway involving mitogen‐activated protein kinases and phosphoinositide 3‐kinases in HeLa cells
Authors: Muscella Antonella , Elia Maria Giovanna , Greco Simona , Storelli Carlo , Marsigliante Santo
Published: March 2003

The effects of P2Y2 purinoceptor activation on c‐Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c‐Fos protein and phosphorylated the extracellular signal‐regulated kinases 1 and 2 (ERK1/2). The P2Y2‐stimulated c‐Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by Gö6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen‐activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3‐kinases (PI3K), LY294002 and wortmannin. When Gö6976 and PD098059, or Gö6976 and wortmannin, were combined there was a totally inhibition of P2Y2‐induced c‐Fos increase. Either U73122 or Gö6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol‐to‐membrane translocation of PKC‐ε induced by ATP/UTP. These data indicated that agonist‐induced PI3K and downstream PKC‐ε activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose‐dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC‐ε pathways to induce c‐Fos protein and HeLa cell proliferation. © 2003 Wiley‐Liss, Inc.
Differential effects of quercetin, a natural polyphenolic flavonoid, on L‐Type calcium current in pituitary tumor (GH3) cells and neuronal NG108‐15 cells
Authors: Wu Sheng‐Nan , Chiang Hung‐Ting , Shen Ai‐Yu , Lo Yuk‐Keung
Published: March 2003

The effects of quercetin, a natural polyphenolic compound, on voltage‐dependent L‐type Ca2+ current (ICa,L) in rat pituitary GH3 cells were investigated with the aid of the whole‐cell voltage‐camp technique. Quercetin (0.5–200 μM) stimulated ICa,L in a concentration‐dependent manner. The current–voltage (I–V) relationship of ICa,L was slightly shifted to more negative potentials in the presence of quercetin. The EC50 value of the quercetin‐induced stimulation of ICa,L was about 7 μM. The presence of quercetin (5 μM) shifted the steady state inactivation curve of ICa,L to a more negative potential by approximately −10 mV. Although quercetin might increase intracellular cyclic AMP, sp‐cAMPS did not affect ICa,L. In addition, neither flavone nor wortmannin had any effect on the amplitude of ICa,L, while epicatechin and genistein slightly suppressed it. Quercetin (50 μM) decreased the amplitude of tetrodotoxin‐sensitive Na+ current in GH3 cells. Under current‐clamp configuration, quercetin could increase the firing frequency of actions potentials. Conversely, in NG108‐15 neuronal cells, quercetin suppressed the amplitude of ICa,L. The quercetin‐induced inhibition of ICa,L was abolished in NG108‐15 cells preincubated with t‐butyl hydroperoxide (1 mM). Quercetin‐mediated stimulation of ICa,L in GH3 cells was presumably not associated with the level of intracellular cyclic AMP, or with the activity of tyrosine or phosphoinositide 3‐kinases. Therefore, the effects of quercetin on ion currents may, at least in part, contribute to the underlying mechanisms through which it affects neuronal or neuroendocrine function. © 2003 Wiley‐Liss, Inc.
Activation of extracellular signal‐regulated kinases ERK1 and ERK2 induces Bcl‐xL up‐regulation via inhibition of caspase activities in erythropoietin signaling
Authors: Mori Masaki , Uchida Mie , Watanabe Tomoko , Kirito Keita , Hatake Kiyohiko , Ozawa Keiya , Komatsu Norio
Published: March 2003

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO‐dependent leukemia cell line UT‐7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT‐7/EPO cells underwent apoptosis, accompanied by down‐regulation of the Bcl‐xL protein. In addition, the cleaved products of caspase‐3, p11 and p21, and a few cleaved forms of inhibitor of caspase‐activated DNase (ICAD) were detected in these cells. When the cells were pre‐treated with the pancaspase inhibitor Z‐VAD‐FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT‐7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal‐regulated kinases (ERKs), the expression of Bcl‐xL protein was down‐regulated and subsequently apoptosis was induced. Interestingly, Z‐VAD‐FMK blocked U0126‐induced down‐regulation of Bcl‐xL protein and apoptosis, strongly suggesting that Bcl‐xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up‐regulates Bcl‐xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis. © 2003 Wiley‐Liss, Inc.
VEGF‐TRAPR1R2 suppresses choroidal neovascularization and VEGF‐induced breakdown of the blood–retinal barrier
Authors: Saishin Yoshitsugu , Saishin Yumiko , Takahashi Kyoichi , Silva Raquel Lima e , Hylton Donna , Rudge John S. , Wiegand Stanley J. , Campochiaro Peter A.
Published: March 2003

Vascular endothelial growth factor (VEGF) plays a central role in the development of retinal neovascularization and diabetic macular edema. There is also evidence suggesting that VEGF is an important stimulator for choroidal neovascularization. In this study, we investigated the effect of a specific inhibitor of VEGF, VEGF‐TRAPR1R2, in models for these disease processes. VEGF‐TRAPR1R2 is a fusion protein, which combines ligand binding elements taken from the extracellular domains of VEGF receptors 1 and 2 fused to the Fc portion of IgG1. Subcutaneous injections or a single intravitreous injection of VEGF‐TRAPR1R2 strongly suppressed choroidal neovascularization in mice with laser‐induced rupture of Bruch's membrane. Subcutaneous injection of VEGF‐TRAPR1R2 also significantly inhibited subretinal neovascularization in transgenic mice that express VEGF in photoreceptors. In two models of VEGF‐induced breakdown of the blood–retinal barrier (BRB), one in which recombinant VEGF is injected into the vitreous cavity and one in which VEGF expression is induced in the retina in transgenic mice, VEGF‐TRAPR1R2 significantly reduced breakdown of the BRB. These data confirm that VEGF is a critical stimulus for the development of choroidal neovascularization and indicate that VEGF‐TRAPR1R2 may provide a new agent for consideration for treatment of patients with choroidal neovascularization and diabetic macular edema. © 2003 Wiley‐Liss, Inc.
Role of protein kinase C α in calcium induced keratinocyte differentiation: Defective regulation in squamous cell carcinoma
Authors: Yang Lee Chuan , Ng Dean C. , Bikle Daniel D.
Published: March 2003

Calcium induces both involucrin and transglutaminase‐K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31‐8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium‐and time‐dependent manner. PKC [epsilon] decreased in a time‐and calcium‐dependent fashion over the three‐week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium‐regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium‐induced differentiation, PKC [alpha] plays a necessary role in mediating calcium‐induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells. © 2003 Wiley‐Liss, Inc.
EGF‐responsive rat neural stem cells: Molecular follow‐up of neuron and astrocyte differentiation in vitro
Authors: Jori F.P. , Galderisi U. , Piegari E. , Cipollaro M. , Cascino A. , Peluso G. , Cotrufo R. , Giordano A. , Melone M.A.B.
Published: March 2003

Neural stem cells (NSCs) could be very useful for the “cell therapy” treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF‐responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF‐containing medium, cells were plated on poly‐L‐ornithine‐coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the “p53 network,” was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation. © 2003 Wiley‐Liss, Inc.
β‐adrenergic receptor‐mediated DNA synthesis in neonatal rat cardiac fibroblasts proceeds via a phosphatidylinositol 3‐kinase dependent pathway refractory to the antiproliferative action of cyclic AMP
Authors: Colombo Federico , Gosselin Hugues , El‐Helou Viviane , Calderone Angelino
Published: March 2003

The following study was undertaken to elucidate the cytoskeletal phenotype of neonatal rat cardiac fibroblasts (NNCF) and the signaling pathways coupled to β‐adrenergic receptor stimulated DNA synthesis. The cytoskeletal proteins vimentin, and smooth muscle α‐actin were detected in NNCF, suggestive of a myofibroblast phenotype. Isoproterenol (ISO) treatment stimulated 3H‐thymidine uptake, and concomitantly increased intracellular cyclic AMP levels. However, cyclic AMP‐elevating agents markedly decreased DNA synthesis. Coincident with growth, ISO‐stimulated phosphatidylinositol 3‐kinase (PI3‐K) activity, and the PI3‐K inhibitor LY294002 abrogated enzyme activity, and DNA synthesis. Unexpectedly, the serine/threonine kinase protein kinase Bα (PKBα), a putative downstream target of PI3‐K, was dephosphorylated following ISO treatment. Despite PKBα inactivation, the phosphorylation of its putative downstream target, the pro‐apoptotic enzyme glycogen synthase kinase‐3α was significantly increased in response to ISO. These latter effects of ISO were mimicked by the cyclic AMP‐elevating agent forskolin. Lastly, ISO treatment increased p70 ribosomal S6 kinase (p70S6K) phosphorylation, as reflected by an upward electrophoretic mobility shift. The pretreatment with rapamycin abrogated the ISO‐mediated mobility shift of p70S6K, and DNA synthesis. Collectively, these data demonstrate that NNCF express a myofibroblast phenotype, and β‐adrenergic agonists promote DNA synthesis via a PI3‐K‐dependent pathway involving p70S6K. Although unable to suppress ISO‐stimulated DNA synthesis, cyclic AMP can influence specific downstream targets of PI3‐K highlighting a novel crosstalk between these signaling pathways. © 2003 Wiley‐Liss, Inc.
RET and NTRK1 proto‐oncogenes in human diseases
Authors: Alberti Luisella , Carniti Cristiana , Miranda Claudia , Roccato Emanuela , Pierotti Marco A.
Published: March 2003

RET and NTRK1 are receptor tyrosine kinase (RTK) proteins which play a role in the development and maturation of specific component of the nervous system. Their alterations have been associated to several human diseases, including some forms of cancer and developmental abnormalities. These features have contributed to the concept that one gene can be responsible for more than one disease. Moreover, both genes encoding for the two RTKs show genetic alterations that belong to either “gain of function” or “loss of function” class of mutations. In fact, receptor rearrangements or point mutations convert RET and NTRK1 in dominantly acting transforming genes leading to thyroid tumors, whereas inactivating mutations, associated with Hirschsprung's disease (HSCR) and congenital insensitivity to pain with anhidrosis (CIPA), impair RET and NTRK1 functions, respectively. In this review we have summarized the main features of the two receptors, their physiological and pathological roles. In addition, we attempted to identify the correlations between the different genetic alterations and the related pathogenetic mechanisms. © 2003 Wiley‐Liss, Inc.